Updated on 2024/04/01

写真a

 
TSUNEMATSU Yuta
 
Organization
Graduate School of Bioagricultural Sciences Department of Applied Biosciences Associate professor
Graduate School
Graduate School of Bioagricultural Sciences
Undergraduate School
School of Agricultural Sciences Department of Applied Biosciences
Title
Associate professor
Contact information
メールアドレス
External link

Degree 1

  1. Ph. D. ( 2011.3   Kyoto University ) 

Research Interests 10

  1. きのこ

  2. 冬虫夏草

  3. Synthetic Biology

  4. colorectal cancer

  5. chemistry of natural products

  6. biosynthesis

  7. 生物有機化学

  8. 生薬学

  9. Aspergillus

  10. 腸内細菌

Research Areas 4

  1. Nanotechnology/Materials / Chemical biology

  2. Life Science / Environmental and natural pharmaceutical resources

  3. Nanotechnology/Materials / Chemistry and chemical methodology of biomolecules

  4. Life Science / Bioorganic chemistry

Research History 11

  1. The University of Tokyo

    2023.4 - 2024.3

  2. National Institute for Environmental Studies

    2022.7

  3. Nagoya University   Graduate School of Bioagricultural Sciences   Associate professor

    2022.4

  4. FY2021, FOREST (Fusion Oriented REsearch for disruptive Science and Technology) program, JST

    2022.4

  5. A start-up company in the USA   Consultant

    2021.4 - 2022.3

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    Country:United States

  6. University of Shizuoka   Department of Pharmaceutical Sciences   Assistant Professor

    2017.7 - 2022.3

  7. Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI)   Biomolecular Chemistry   Postdoctral researcher

    2014.9 - 2015.8

  8. University of Shizuoka   Department of Pharmaceutical Sciences   Research Assistant Professor

    2014.8 - 2017.6

  9. University of Shizuoka   Department of Pharmaceutical Sciences   Designated assistant professor

    2012.4 - 2014.7

  10. University of Shizuoka   Department of Pharmaceutical Sciences   JSPS fellow (PD)

    2011.4 - 2012.3

  11. Kyoto University   Department of Pharmaceutical Sciences   JSPS fellow (DC2)

    2010.4 - 2011.3

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Education 2

  1. Kyoto University   Department of Pharmaceutical Sciences

    2008.4 - 2011.3

  2. Nagoya University   Department of Chemistry

    2006.4 - 2008.3

Professional Memberships 6

  1. SOCIETY FOR ACTINOMYCETES JAPAN

  2. The Society for Biotechnology, Japan

  3. THE JAPANESE SOCIETY OF PHARMACOGNOSY

  4. THE PHARMACEUTICAL SOCIETY OF JAPAN

  5. JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY

  6. 糸状菌分子生物学研究会

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Committee Memberships 3

  1. 日本農芸化学会   2018年度大会プログラム編成会議招聘メンバー  

    2017.12   

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    Committee type:Academic society

  2. 天然物化学談話会   世話人  

    2017.4   

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    Committee type:Academic society

  3. 静岡県立大学薬学部   研究倫理審査部局委員  

    2017.4 - 2022.3   

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    Committee type:Academic society

Awards 8

  1. The PSJ Award for Young Scientists

    2019.9   The Japanese Society of Pharmacognosy   Decrypting and harnessing biosynthetic potential of natural products in fungal genome

    Yuta Tsunematsu

  2. Best Presentation Award

    2017.6   The 24th Auual Meeting of the Japanese Association for Cancer Prevention  

    Yuta Tsunematsu

  3. Best Presentation Award

    2016.6   The Japanese Association for the Pursuit of New Bioactive Resources  

    Yuta Tsunematsu

  4. Best Poster Award

    2016.3   Biosystems Design 2.0 Symposium  

    Yuta Tsunematsu

  5. Young Scientist's Research Award in Natural Product Chemistry

    2014.10  

    Yuta Tsunematsu

  6. Young Scientist Research Award

    2013.10   The Pharmaceutical Society of Japan/Division of Natural Medicines  

    Yuta Tsunematsu

  7. 10th Award for Leave a Nest Grants

    2012.4   Leave a Nest Co., ltd.  

    Yuta Tsunematsu

  8. Japan Young Scientist Research Award in Natural Product Chemistry

    2010.10  

    Yuta Tsunematsu

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Papers 71

  1. Specialized Flavoprotein Promotes Sulfur Migration and Spiroaminal Formation in Aspirochlorine Biosynthesis. Reviewed International journal

    Yuta Tsunematsu, Naoya Maeda, Michio Sato, Kodai Hara, Hiroshi Hashimoto, Kenji Watanabe, Christian Hertweck

    Journal of the American Chemical Society   Vol. 143 ( 1 ) page: 206 - 213   2021.1

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    Epidithiodiketopiperazines (ETPs) are a class of ecologically and medicinally important cyclodipeptides bearing a reactive transannular disulfide bridge. Aspirochlorine, an antifungal and toxic ETP isolated from Aspergillus oryzae used in sake brewing, deviates from the common ETP scaffold owing to its unusual ring-enlarged disulfide bridge linked to a spiroaminal ring system. Although this disulfide ring system is implicated in the biological activity of ETPs the biochemical basis for this derailment has remained a mystery. Here we report the discovery of a novel oxidoreductase (AclR) that represents the first-in-class enzyme catalyzing both a carbon-sulfur bond migration and spiro-ring formation, and that the acl pathway involves a cryptic acetylation as a prerequisite for the rearrangement. Genetic screening in A. oryzae identified aclR as the candidate for the complex biotransformation, and the aclR-deficient mutant provided the biosynthetic intermediate, unexpectedly harboring an acetyl group. In vitro assays showed that AclR alone promotes 1,2-sulfamyl migration, elimination of the acetoxy group, and spiroaminal formation. AclR features a thioredoxin oxidoreductase fold with a noncanonical CXXH motif that is distinct from the CXXC in the disulfide forming oxidase for the ETP biosynthesis. Crystallographic and mutational analyses of AclR revealed that the CXXH motif is crucial for catalysis, whereas the flavin-adenine dinucleotide is required as a support of the protein fold, and not as a redox cofactor. AclR proved to be a suitable bioinformatics handle to discover a number of related fungal gene clusters that potentially code for the biosynthesis of derailed ETP compounds. Our results highlight a specialized role of the thioredoxin oxidoreductase family enzyme in the ETP pathway and expand the chemical diversity of small molecules bearing an aberrant disulfide pharmacophore.

    DOI: 10.1021/jacs.0c08879

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  2. Genomic Mushroom Hunting Decrypts Coprinoferrin, A Siderophore Secondary Metabolite Vital to Fungal Cell Development. Reviewed

    Tsunematsu Y, Takanishi J, Asai S, Masuya T, Nakazawa T, Watanabe K

    Organic letters   Vol. 21 ( 18 ) page: 7582 - 7586   2019.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/acs.orglett.9b02861

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  3. Enzymatic Amide Tailoring Promotes Retro-Aldol Amino Acid Conversion to Form the Antifungal Agent Aspirochlorine. Reviewed

    Tsunematsu Y, Maeda N, Yokoyama M, Chankhamjon P, Watanabe K, Scherlach K, Hertweck C

    Angewandte Chemie (International ed. in English)   Vol. 57 ( 43 ) page: 14051 - 14054   2018.10

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  4. Oxidative trans to cis Isomerization of Olefins in Polyketide Biosynthesis Reviewed

    Tsuyoshi Yamamoto, Yuta Tsunematsu, Kodai Hara, Tomohiro Suzuki, Shinji Kishimoto, Hirokazu Kawagishi, Hiroshi Noguchi, Hiroshi Hashimoto, Yi Tang, Kinya Hotta, Kenji Watanabe

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   Vol. 55 ( 21 ) page: 6207 - 10   2016.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

    Geometric isomerization can expand the scope of biological activities of natural products. The observed chemical diversity among the pseurotin-type fungal secondary metabolites is in part generated by a trans to cis isomerization of an olefin. In vitro characterizations of pseurotin biosynthetic enzymes revealed that the glutathione S-transferase PsoE requires participation of the bifunctional C-methyltransferase/epoxidase PsoF to complete the trans to cis isomerization of the pathway intermediate presynerazol. The crystal structure of the PsoE/ glutathione/ presynerazol complex indicated stereospecific glutathione-presynerazol conjugate formation is the principal function of PsoE. Moreover, PsoF was identified to have an additional, unexpected oxidative isomerase activity, thus making it a trifunctional enzyme which is key to the complexity generation in pseurotin biosynthesis. Through the study, we identified a novel mechanism of accomplishing a seemingly simple trans to cis isomerization reaction.

    DOI: 10.1002/anie.201600940

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  5. Isolation, Structure Elucidation, and Total Synthesis of Tryptopeptins A and B, New TGF-beta Signaling Modulators from Streptomyces sp. Reviewed

    Yuta Tsunematsu, Shinichi Nishimura, Akira Hattori, Shinya Oishi, Nobutaka Fujii, Hideaki Kakeya

    ORGANIC LETTERS   Vol. 17 ( 2 ) page: 258 - 61   2015.1

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    Two new microbial metabolites, tryptopeptins A (1) and B (2), were isolated from the cultured broth of Streptomyces sp. KUSC-G11, as modulators of the transforming growth factor-beta (TGF-beta) signaling pathway. Their chemical structures consisting of isovalerate, N-Me-l-Val, l-allo-Thr, and a tryptophan-related residue were elucidated on the basis of spectroscopic analyses, while they were unambiguously determined by total syntheses. A structureactivity relationship (SAR) study using natural and synthesized tryptopeptins revealed the importance of the alpha,beta-epoxyketone function located at the C terminus. These new TGF-beta signaling modulators would be highly useful for development of new drug leads targeting TGF-beta-related diseases such as fibrosis and cancer.

    DOI: 10.1021/ol503340k

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  6. Elucidation of Pseurotin Biosynthetic Pathway Points to Trans-Acting C-Methyltransferase: Generation of Chemical Diversity Reviewed

    Yuta Tsunematsu, Manami Fukutomi, Takayoshi Saruwatari, Hiroshi Noguchi, Kinya Hotta, Yi Tang, Kenji Watanabe

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   Vol. 53 ( 32 ) page: 8475 - 9   2014.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-V C H VERLAG GMBH  

    Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivity. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. Systematic deletion of the pseurotin biosynthetic genes in A. fumigatus and in vivo and in vitro characterization of the tailoring enzymes to determine the biosynthetic intermediates, and the gene products responsible for the formation of each intermediate, are described. Thus, the main biosynthetic steps leading to the formation of pseurotin A from the predominant precursor, azaspirene, were elucidated. The study revealed the combinatorial nature of the biosynthesis of the pseurotin family of compounds and the intermediates. Most interestingly, we report the first identification of an epoxidase C-methyltransferase bifunctional fusion protein PsoF which appears to methylate the nascent polyketide backbone carbon atom in trans.

    DOI: 10.1002/anie.201404804

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  7. 1,2,3-Triazine formation mechanism of the fairy chemical 2-azahypoxanthine in the fairy ring-forming fungus Lepista sordida. International journal

    Akinobu Ito, Jae-Hoon Choi, Waki Yokoyama-Maruyama, Mihaya Kotajima, Jing Wu, Tomohiro Suzuki, Yurika Terashima, Hyogo Suzuki, Hirofumi Hirai, David C Nelson, Yuta Tsunematsu, Kenji Watanabe, Tomohiro Asakawa, Hitoshi Ouchi, Makoto Inai, Hideo Dohra, Hirokazu Kawagishi

    Organic & biomolecular chemistry   Vol. 20 ( 13 ) page: 2636 - 2642   2022.3

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    2-Azahypoxanthine (AHX) was first isolated from the culture broth of the fungus Lepista sordida as a fairy ring-inducing compound. It has since been found that a large number of plants and mushrooms produce AHX endogenously and that AHX has beneficial effects on plant growth. The AHX molecule has an unusual, nitrogen-rich 1,2,3-triazine moiety of unknown biosynthetic origin. Here, we establish the biosynthetic pathway for AHX formation in L. sordida. Our results reveal that the key nitrogen sources that are responsible for the 1,2,3-triazine formation are reactive nitrogen species (RNS), which are derived from nitric oxide (NO) produced by NO synthase (NOS). Furthermore, RNS are also involved in the biochemical conversion of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (AICAR) to AHX-ribotide (AHXR), suggesting that a novel biosynthetic route that produces AHX exists in the fungus. These findings demonstrate a physiological role for NOS in AHX biosynthesis as well as in biosynthesis of other natural products containing a nitrogen-nitrogen bond.

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  8. Induction of DNA Damage in Mouse Colorectum by Administration of Colibactin-producing <i>Escherichia coli</i>, Isolated from a Patient With Colorectal Cancer

    TAKUMI NARITA, YUTA TSUNEMATSU, NORIYUKI MIYOSHI, MASAMI KOMIYA, TAKAHIRO HAMOYA, GEN FUJII, YUKO YOSHIKAWA, MICHIO SATO, MASANOBU KAWANISHI, HARUHIKO SUGIMURA, YUJI IWASHITA, YUKARI TOTSUKA, MASARU TERASAKI, KENJI WATANABE, KEIJI WAKABAYASHI, MICHIHIRO MUTOH

    In Vivo   Vol. 36 ( 2 ) page: 628 - 634   2022.3

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    DOI: 10.21873/invivo.12746

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  9. Toward Engineered Biosynthesis of Drugs in Human Cells. International journal

    Shinya Matsuda, Yuta Tsunematsu, Takuma Matsushita, Yuji Ogata, Shihomi Hachiya, Shinji Kishimoto, Noriyuki Miyoshi, Kenji Watanabe

    Chembiochem : a European journal of chemical biology   Vol. 23 ( 4 ) page: e202100645   2022.2

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    Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.

    DOI: 10.1002/cbic.202100645

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  10. 日本人における身体活動量とコリバクチン産生菌の関連

    二宮 友佳, 中潟 崇, 南里 妃名子, 大野 治美, 谷澤 薫平, 小西 可奈, 村上 晴香, 恒松 雄太, 佐藤 道大, 渡辺 賢二, 宮地 元彦

    健康支援   Vol. 24 ( 1 ) page: 118 - 118   2022.2

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  11. Association of Escherichia coli containing polyketide synthase in the gut microbiota with colorectal neoplasia in Japan. International journal

    Motoki Iwasaki, Rieko Kanehara, Taiki Yamaji, Ryoko Katagiri, Michihiro Mutoh, Yuta Tsunematsu, Michio Sato, Kenji Watanabe, Koji Hosomi, Yasuo Kakugawa, Hiroaki Ikematsu, Kinichi Hotta, Jun Kunisawa, Keiji Wakabayashi, Takahisa Matsuda

    Cancer science   Vol. 113 ( 1 ) page: 277 - 286   2022.1

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    Escherichia coli containing polyketide synthase in the gut microbiota (pks+ E coli) produce a polyketide-peptide genotoxin, colibactin, and are suspected to play a role in the development of colorectal neoplasia. To clarify the role of pks+ E coli in the early stage of tumorigenesis, we investigated whether the pks status of E coli was associated with the prevalence of colorectal neoplasia. This cross-sectional analysis of data from a prospective cohort in Izu Oshima, Japan included asymptomatic residents aged 40-79 years who underwent screening colonoscopy and provided a stool sample. We identified 543 participants with colorectal neoplasia (22 colorectal cancer and 521 adenoma) as cases and 425 participants with normal colon as controls. The pks status of E coli was assayed using stool DNA and specific primers that detected pks+ E coli. The proportion of pks+ E coli was 32.6% among cases and 30.8% among controls. Compared with those with pks- E coli, the odds ratio (OR) (95% confidence interval) for participants with pks+ E coli was 1.04 (0.77-1.41) after adjusting for potential confounders. No statistically significant associations were observed regardless of tumor site or number of colorectal adenoma lesions. However, stratified analyses revealed increased ORs among participants who consumed cereals over the median intake or vegetables under the median intake. Overall, we found no statistically significant association between pks+ E coli and the prevalence of colorectal adenoma lesions among this Japanese cohort. However, positive associations were suggested under certain intake levels of cereals or vegetables.

    DOI: 10.1111/cas.15196

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  12. 担子菌休眠型生合成遺伝子の覚醒による新規天然物の発見とその菌糸成長・子実体形成作用 Reviewed

    恒松 雄太

    アグリバイオ = Agricultural biotechnology   Vol. 5 ( 12 ) page: 1063 - 1067   2021.11

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    Language:Japanese   Publisher:東京 : 北隆館  

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  13. Mother-to-infant transmission of the carcinogenic colibactin-producing bacteria. Reviewed International journal

    Yuta Tsunematsu, Koji Hosomi, Jun Kunisawa, Michio Sato, Noriko Shibuya, Emiko Saito, Haruka Murakami, Yuko Yoshikawa, Yuji Iwashita, Noriyuki Miyoshi, Michihiro Mutoh, Hideki Ishikawa, Haruhiko Sugimura, Motohiko Miyachi, Keiji Wakabayashi, Kenji Watanabe

    BMC microbiology   Vol. 21 ( 1 ) page: 235 - 235   2021.8

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    BACKGROUND: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized. RESULTS: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake. CONCLUSIONS: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer.

    DOI: 10.1186/s12866-021-02292-1

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  14. Stool pattern is associated with not only the prevalence of tumorigenic bacteria isolated from fecal matter but also plasma and fecal fatty acids in healthy Japanese adults. International journal

    Daiki Watanabe, Haruka Murakami, Harumi Ohno, Kumpei Tanisawa, Kana Konishi, Kikue Todoroki-Mori, Yuta Tsunematsu, Michio Sato, Yuji Ogata, Noriyuki Miyoshi, Naoto Kubota, Jun Kunisawa, Keiji Wakabayashi, Tetsuya Kubota, Kenji Watanabe, Motohiko Miyachi

    BMC microbiology   Vol. 21 ( 1 ) page: 196 - 196   2021.6

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    BACKGROUND: Colibactin-producing Escherichia coli containing polyketide synthase (pks+ E. coli) has been shown to be involved in colorectal cancer (CRC) development through gut microbiota analysis in animal models. Stool status has been associated with potentially adverse gut microbiome profiles from fecal analysis in adults. We examined the association between stool patterns and the prevalence of pks+ E. coli isolated from microbiota in fecal samples of 224 healthy Japanese individuals. RESULTS: Stool patterns were determined through factorial analysis using a previously validated questionnaire that included stool frequency, volume, color, shape, and odor. Factor scores were classified into tertiles. The prevalence of pks+ E. coli was determined by using specific primers for pks+ E. coli in fecal samples. Plasma and fecal fatty acids were measured via gas chromatography-mass spectrometry. The prevalence of pks+ E. coli was 26.8%. Three stool patterns identified by factorial analysis accounted for 70.1% of all patterns seen (factor 1: lower frequency, darker color, and harder shape; factor 2: higher volume and softer shape; and factor 3: darker color and stronger odor). Multivariable-adjusted odds ratios (95% confidence intervals) of the prevalence of pks+ E. coli for the highest versus the lowest third of the factor 1 score was 3.16 (1.38 to 7.24; P for trend = 0.006). This stool pattern exhibited a significant positive correlation with fecal isobutyrate, isovalerate, valerate, and hexanoate but showed a significant negative correlation with plasma eicosenoic acid and α-linoleic acid, as well as fecal propionate and succinate. No other stool patterns were significant. CONCLUSIONS: These results suggest that stool patterns may be useful in the evaluation of the presence of tumorigenic bacteria and fecal fatty acids through self-monitoring of stool status without the requirement for specialist technology or skill. Furthermore, it may provide valuable insight about effective strategies for the early discovery of CRC.

    DOI: 10.1186/s12866-021-02255-6

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  15. Isolation of New Colibactin Metabolites from Wild-Type Escherichia coli and In Situ Trapping of a Mature Colibactin Derivative. Reviewed International journal

    Tao Zhou, Yuichiro Hirayama, Yuta Tsunematsu, Nanami Suzuki, Seiji Tanaka, Nahoko Uchiyama, Yukihiro Goda, Yuko Yoshikawa, Yuji Iwashita, Michio Sato, Noriyuki Miyoshi, Michihiro Mutoh, Hideki Ishikawa, Haruhiko Sugimura, Keiji Wakabayashi, Kenji Watanabe

    Journal of the American Chemical Society   Vol. 143 ( 14 ) page: 5526 - 5533   2021.4

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    Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

    DOI: 10.1021/jacs.1c01495

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  16. o-Anisidine Dimer, 2-Methoxy-N4-(2-methoxyphenyl) Benzene-1,4-diamine, in Rat Urine Associated with Urinary bladder Carcinogenesis. Reviewed International journal

    Takuma Kobayashi, Takeshi Toyoda, Yuya Tajima, Shinji Kishimoto, Yuta Tsunematsu, Michio Sato, Kohei Matsushita, Takanori Yamada, Yuko Shimamura, Shuichi Masuda, Masako Ochiai, Kumiko Ogawa, Kenji Watanabe, Takeji Takamura-Enya, Yukari Totsuka, Keiji Wakabayashi, Noriyuki Miyoshi

    Chemical research in toxicology   Vol. 34 ( 3 ) page: 912 - 919   2021.3

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    Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

    DOI: 10.1021/acs.chemrestox.0c00536

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  17. Genomics-directed activation of cryptic natural product pathways deciphers codes for biosynthesis and molecular function. Reviewed

    Yuta Tsunematsu

    Journal of natural medicines   Vol. 75 ( 2 ) page: 261 - 274   2021.3

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    Natural products, which can be isolated from living organisms worldwide, have played a pivotal role in drug discovery since ancient times. However, it has become more challenging to identify a structurally novel molecule with promising biological activity for pharmaceutical development, mainly due to the limited methodologies for their acquisition. In this review, we summarize our recent studies that activate the biosynthetic potential of filamentous fungi by genetic engineering to harness the metabolic flow for the efficient production of unprecedented natural products. The recent revolution in genome sequencing technology enables the accumulation of vast amounts of information on biosynthetic genes, the blueprint of the molecular construction. Utilizing the established heterologous expression system, activation of the pathway-specific transcription factor coupled with a knockout strategy, and manipulating the global regulatory gene, the biosynthetic genes were exploited to activate biosynthetic pathways and decipher the encoded enzyme functions. We show that this methodology was beneficial for acquiring fungal treasures for drug discovery. These studies also enabled the investigation of the molecular function of natural products in fungal development.

    DOI: 10.1007/s11418-020-01466-x

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  18. Catalytic mechanism and endo-to-exo selectivity reversion of an octalin-forming natural Diels-Alderase

    Michio Sato, Shinji Kishimoto, Mamoru Yokoyama, Cooper S. Jamieson, Kazuto Narita, Naoya Maeda, Kodai Hara, Hiroshi Hashimoto, Yuta Tsunematsu, Kendall N. Houk, Yi Tang, Kenji Watanabe

    NATURE CATALYSIS   Vol. 4 ( 3 ) page: 223 - 232   2021.3

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    We have previously reported the identification of CghA, a proposed Diels-Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA-product complex at a resolution of 2.0 angstrom. Our result provides the second structural determination of eukaryotic Diels-Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.

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  19. Characterization of colibactin-producing Escherichia coli isolated from Japanese patients with colorectal cancer. Reviewed

    Yuko Yoshikawa, Yuta Tsunematsu, Nobuo Matsuzaki, Yuichiro Hirayama, Fumi Higashiguchi, Michio Sato, Yuji Iwashita, Noriyuki Miyoshi, Michihiro Mutoh, Hideki Ishikawa, Haruhiko Sugimura, Keiji Wakabayashi, Kenji Watanabe

    Japanese journal of infectious diseases   Vol. 73 ( 6 ) page: 437 - 442   2020.11

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    We investigated the relationship between colibactin-producing (clb+) Escherichia coli and colorectal adenocarcinoma. In total, 729 E. coli colonies were isolated from tumor and surrounding non-tumor regions in resected specimens from 34 Japanese patients; 450 colonies were from tumor regions and 279 from non-tumor regions. clb+ bacteria were found in tumor regions of 11 patients (11/34, 32.4%) and in non-tumor regions of seven of the 11 (7/34, 20.6%). The prevalence of clb+ isolates was 72.7% (327/450) and 44.1% (123/279) in tumor and non-tumor regions, respectively. All the recovered clb+ isolates belonged to the phylogenetic group B2 and were the most predominant type in tumor regions. Hemolytic (α-hemolysin-positive, hlyA+) and non-hemolytic (α-hemolysin-negative, hlyA-) clb+ isolates were obtained from patient #19; however, the prevalence of hlyA+ clb+ isolates was significantly higher in tumor regions (35/43, 81.4%) than in non-tumor regions (3/19, 15.8%). Moreover, a significantly higher production of N-myristoyl-D-asparagine, a byproduct of colibactin biosynthesis, was observed in hlyA+ clb+ isolates than in hlyA- clb+ isolates. Our results suggest that hlyA+ clb+ E. coli may have a selective advantage in colorectal colonization, consequently playing a role in carcinogenesis. The presence of hlyA+ clb+ bacteria in healthy individuals is a risk marker of colorectal cancer.

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  20. Uncovering hidden sesquiterpene biosynthetic pathway through expression boost area-mediated productivity enhancement in basidiomycete. Reviewed

    Shihori Asai, Yuta Tsunematsu, Takahiro Masuya, Junnosuke Otaka, Hiroyuki Osada, Kenji Watanabe

    The Journal of antibiotics   Vol. 73 ( 10 ) page: 721 - 728   2020.10

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    Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin-hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.

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  21. Association between dietary intake and the prevalence of tumourigenic bacteria in the gut microbiota of middle-aged Japanese adults. Reviewed International journal

    Daiki Watanabe, Haruka Murakami, Harumi Ohno, Kumpei Tanisawa, Kana Konishi, Yuta Tsunematsu, Michio Sato, Noriyuki Miyoshi, Keiji Wakabayashi, Kenji Watanabe, Motohiko Miyachi

    Scientific reports   Vol. 10 ( 1 ) page: 15221 - 15221   2020.9

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    The relative contribution of diet to colorectal cancer (CRC) incidence is higher than that for other cancers. Animal models have revealed that Escherichia coli containing polyketide synthase (pks+ E. coli) in the gut participates in CRC development. The purpose of this cross-sectional study was to examine the relationship between dietary intake and the prevalence of pks+ E. coli isolated from the microbiota in faecal samples of 223 healthy Japanese individuals. Dietary intake was assessed using a previously validated brief-type self-administered diet history questionnaire. The prevalence of pks+ E. coli was evaluated using faecal samples collected from participants and specific primers that detected pks+ E. coli. The prevalence of pks+ E. coli was 26.9%. After adjusting for baseline confounders, the prevalence of pks+ E. coli was negatively associated with the intake of green tea (odds ratio [OR], 0.59 [95% confidence interval (CI) 0.30-0.88] per 100 g/1,000 kcal increment) and manganese (OR, 0.43 [95% CI 0.22-0.85] per 1 mg/1,000 kcal increment) and was positively associated with male sex (OR, 2.27 [95% CI 1.05-4.91]). While futher studies are needed to validate these findings, these results provide insight into potential dietary interventions for the prevention of CRC.

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  22. Elevated levels of proinflammatory volatile metabolites in feces of high fat diet fed KK-Ay mice. Reviewed International journal

    Misaki Uchikawa, Mai Kato, Akika Nagata, Shunsuke Sanada, Yuto Yoshikawa, Yuta Tsunematsu, Michio Sato, Takuji Suzuki, Tsutomu Hashidume, Kenji Watanabe, Yuko Yoshikawa, Noriyuki Miyoshi

    Scientific reports   Vol. 10 ( 1 ) page: 5681 - 5681   2020.3

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    When the microfloral composition deteriorates, it triggers low-level chronic inflammation associated with several lifestyle-related diseases including obesity and diabetic mellitus. Fecal volatile organic compounds (VOCs) have been found to differ in gastrointestinal diseases as well as intestinal infection. In this study, to evaluate a potential association between the pathogenesis of lifestyle-related diseases and VOCs in the intestinal tract, fecal VOCs from obese/diabetic KK-Ay mice (KK) or controls (C57BL/6J mice; BL) fed a normal or high fat diet (NFD or HFD) were investigated using headspace sampler-GC-EI-MS. Principal component analysis (PCA) of fecal VOC profiles clearly separated the experimental groups depending on the mouse lineage (KK vs BL) and the diet type (NFD vs HFD). 16 s rRNA sequencing revealed that the PCA distribution of VOCs was in parallel with the microfloral composition. We identified that some volatile metabolites including n-alkanals (nonanal and octanal), acetone and phenol were significantly increased in the HFD and/or KK groups. Additionally, these volatile metabolites induced proinflammatory activity in the RAW264 murine macrophage cell line indicating these bioactive metabolites might trigger low-level chronic inflammation. These results suggest that proinflammatory VOCs detected in HFD-fed and/or diabetic model mice might be novel noninvasive diagnosis biomarkers for diabetes.

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  23. Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms. Reviewed International journal

    Masanobu Kawanishi, Chiaki Shimohara, Yoshimitsu Oda, Yuuta Hisatomi, Yuta Tsunematsu, Michio Sato, Yuichiro Hirayama, Noriyuki Miyoshi, Yuji Iwashita, Yuko Yoshikawa, Haruhiko Sugimura, Michihiro Mutoh, Hideki Ishikawa, Keiji Wakabayashi, Takashi Yagi, Kenji Watanabe

    Genes and environment : the official journal of the Japanese Environmental Mutagen Society   Vol. 42 ( Article number: 12 ) page: 12 - 12   2020

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    Introduction: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb+) microorganisms possess a 54-kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. Findings: The analysis revealed that two of six strains possessed a clb gene cluster. These clb+ strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. Conclusion: These results support the observations that the clb gene cluster is widely distributed in nature and clb+E. coli having genotoxic potencies is not rare among microorganisms.

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  24. Diastereoselectivity and product inhibition evasion by Diels-Alderase

    Sato M., Kishimoto S., Jamieson C. S., Hara K., Hashimoto H., Tsunematsu Y., Kendall H. N., Tang Y., Watanabe K.

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 62 ( 0 ) page: 181-186   2020

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  25. Discovery of coprinoferrin isolated from a Basidiomycete for the development of fruiting body

    Takanishi J., Tsunematsu Y., Masuya T., Isogai T., Hayakawa I., Sakakura A., Watanabe K.

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 62 ( 0 ) page: 391-396   2020

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  26. Functional and Structural Analyses of trans C-Methyltransferase in Fungal Polyketide Biosynthesis. Reviewed International journal

    Shinji Kishimoto, Yuta Tsunematsu, Takuma Matsushita, Kodai Hara, Hiroshi Hashimoto, Yi Tang, Kenji Watanabe

    Biochemistry   Vol. 58 ( 38 ) page: 3933 - 3937   2019.9

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    Biosynthesis of certain fungal polyketide-peptide synthetases involves C-methyltransferase activity that adds one or more S-adenosyl-l-methionine-derived methyl groups to the carbon framework. The previously reported PsoF-MT, the stand-alone C-methyltransferase (MT) from the pseurotin biosynthetic pathway that exists as a domain within a trifunctional didomain enzyme PsoF, was characterized crystallographically and kinetically using mutants with substrate analogs to understand how a trans-acting C-MT works and compare it to known polyketide synthase-associated C-MTs. This study identified key active-site residues involved in catalysis and substrate recognition, which led us to propose the mechanism of C-methylation and substrate specificity determinants in PsoF-MT.

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  27. Activity-Based Probe for Screening of High-Colibactin Producers from Clinical Samples. Reviewed International journal

    Yuichiro Hirayama, Yuta Tsunematsu, Yuko Yoshikawa, Ryota Tamafune, Nobuo Matsuzaki, Yuji Iwashita, Ippei Ohnishi, Fumihiko Tanioka, Michio Sato, Noriyuki Miyoshi, Michihiro Mutoh, Hideki Ishikawa, Haruhiko Sugimura, Keiji Wakabayashi, Kenji Watanabe

    Organic letters   Vol. 21 ( 12 ) page: 4490 - 4494   2019.6

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    While high-colibactin-producing Escherichia coli is thought to be associated with colorectal oncogenesis, this study is complicated part due to an inability to isolate colibactin adequately. Here, we created fluorescent probes activated by ClbP, the colibactin-maturing peptidase, to identify high-colibactin-producing strains. Our probe served as a valuable clinical diagnostic tool that allowed simple high-throughput diagnostic screening of clinical samples. Furthermore, the probe also allowed identification of high-colibactin producers that would help advance our understanding of colibactin biosynthesis.

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  28. Biosynthesis of lagopodins in mushroom involves a complex network of oxidation reactions. Reviewed

    Masuya T, Tsunematsu Y, Hirayama Y, Sato M, Noguchi H, Nakazawa T, Watanabe K

    Organic & biomolecular chemistry   Vol. 17 ( 2 ) page: 234 - 239   2019.1

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    <p>Targeted gene knockout in <italic>Coprinopsis cinerea</italic>, yeast <italic>in vivo</italic> bioconversion and <italic>in vitro</italic> assays elucidated the lagopodin biosynthetic pathway, including a complexity-generating network of oxidation steps.</p>

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  29. In vitro genotoxicity analyses of colibactin-producing E. coli isolated from a Japanese colorectal cancer patient. Reviewed

    Masanobu Kawanishi, Yuuta Hisatomi, Yoshimitsu Oda, Chiaki Shimohara, Yuta Tsunematsu, Michio Sato, Yuichiro Hirayama, Noriyuki Miyoshi, Yuji Iwashita, Yuko Yoshikawa, Haruhiko Sugimura, Michihiro Mutoh, Hideki Ishikawa, Keiji Wakabayashi, Takashi Yagi, Kenji Watanabe

    The Journal of toxicological sciences   Vol. 44 ( 12 ) page: 871 - 876   2019

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    Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

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  30. Biosynthesis-assisted structure elucidation of colibactin, the genotoxic metabolite produced by commensal microbiota Reviewed

    Yuta Tsunematsu

    Yuki Gosei Kagaku Kyokaishi/Journal of Synthetic Organic Chemistry   Vol. 76 ( 5 ) page: 490 - 493   2018

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    Colibactin, a secondary metabolite produced by a commensal Escherichia coli harboring pks island, exhibits a genotoxicity to mammalian cells and progresses to colorectal cancer. Regardless of potential risk for the promotion of cancer by colibactin, which shows neither a biological mechanism of action nor determination of chemical structure. Analysis of a genetic mutant of colibactin-producing E. coli enabled us to provide biosynthetic intermediates and shunt products associated with a partial structure of colibactin. This review covers the recent findings of the biosynthesis and implication of chemical structure of colibactin.

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  31. 7. Structure and Mechanism of Cyclopenase, a Fungal Enzyme Forming Quinolone Core of Viridicatin-Type Compounds

    Kishimoto Shinji, Hara Kodai, Ishikawa Noriyasu, Yamada Haruka, Tsunematsu Yuta, Hashimoto Hiroshi, Tang Yi, Houk Kendall N., Watanabe Kenji

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 60 ( 0 ) page: 37-42   2018

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  32. 新規疾患治療法開発のヒントは「微生物の生存戦略」に隠されている? Reviewed

    恒松 雄太

    ファルマシア   Vol. 54 ( 5 ) page: 458 - 458   2018

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    冬虫夏草という語を初めて目にし「虫なのか草なのか,一体これは何者だろう?」と興味を抱く人は少なくない.昆虫の亡骸からキノコが生えた奇妙な姿形や,その正体が糸状菌(いわゆるカビ)である事実に驚かされたのは筆者だけではないだろう.冬虫夏草の一種であるサナギタケ<i>Cordyceps</i> <i>militaris</i>は,平板培地においてもキノコを形成するため人工栽培法が確立され,国内でも健康食品として市販されている.その成分の1つがコルジセピン(cordycepin:COR)であり,抗がん,免疫賦活,精力増強など,様々な薬理活性を示すことが知られている.CORはアデノシンの3́位デオキシ体であり,アデノシンとの類似性が故にポリメラーゼによる伸長途中のRNA鎖に取り込まれる.ところが,3́位水酸基が欠如しているため更なる伸長反応が起こらず,細胞増殖が阻害される.一方,CORは生体内のアデニンデアミナーゼ(adenine deaminase:ADA)によって容易に薬物代謝(脱アミノ化)され,活性の減弱化した3́-デオキシイノシン(3́-deoxyinosine:3́dI)を生成する.そのため,かつて実施された白血病治療薬としての臨床試験においては,ADA阻害薬であるペントスタチン(pentostatin:PTN)との併用療法が検討されていた.今回,未解明であったCOR生合成遺伝子が同定され,その独特な生合成戦略が明らかにされたので紹介する.<br>なお,本稿は下記の文献に基づいて,その研究成果を紹介するものである.<br>1) Tuli H. S. <i>et</i> <i>al</i>., <i>Life</i> <i>Sci</i>., <b>93</b>, 863-869(2013).<br>2) Xia Y. <i>et</i> <i>al</i>., <i>Cell</i> <i>Chem</i>. <i>Biol</i>., <b>24</b>, 1479-1489(2017).<br>3) Wu P. <i>et</i> <i>al</i>., <i>Cell</i> <i>Chem</i>. <i>Biol</i>., <b>24</b>, 171-181(2017).

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  33. Effects of pex1 disruption on wood lignin biodegradation, fruiting development and the utilization of carbon sources in the white-rot Agaricomycete Pleurotus ostreatus and non-wood decaying Coprinopsis cinerea Reviewed

    Takehito Nakazawa, Ayako Izuno, Masato Horii, Rina Kodera, Hiroshi Nishimura, Yuichiro Hirayama, Yuta Tsunematsu, Yasumasa Miyazaki, Tatsuya Awano, Hajime Muraguchi, Kenji Watanabe, Masahiro Sakamoto, Keiji Takabe, Takashi Watanabe, Yuji Isagi, Yoichi Honda

    FUNGAL GENETICS AND BIOLOGY   Vol. 109   page: 7 - 15   2017.12

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    Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of peal disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that peal is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous a cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

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  34. Elucidation of Biosynthetic Pathways of Natural Products Reviewed

    Shinji Kishimoto, Yuta Tsunematsu, Michio Sato, Kenji Watanabe

    CHEMICAL RECORD   Vol. 17 ( 11 ) page: 1095 - 1108   2017.11

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    During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.)

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  35. Integration of Chemical, Genetic, and Bioinformatic Approaches Delineates Fungal Polyketide-Peptide Hybrid Biosynthesis Reviewed

    Mamoru Yokoyama, Yuichiro Hirayama, Tsuyoshi Yamamoto, Shinji Kishimoto, Yuta Tsunematsu, Kenji Watanabe

    ORGANIC LETTERS   Vol. 19 ( 8 ) page: 2002 - 2005   2017.4

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    To identify natural products and their associated biosynthetic genes from underutilized, difficult-to-manipulate microbes, chemical screening and bioinformatic analysis were employed to identify secondary metabolites and a potentially associated biosynthetic gene cluster. Subsequently, a heterologous expression system was used to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. This approach successfully identified the curvupallide and spirostaphylotrichin biosynthetic pathways in endophytic fungus Curvularia pallescens and the short-chain pyranonigrin biosynthetic pathway in Aspergillus niger.

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  36. Regioselective Dichlorination of a Non-Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme Reviewed

    Pranatchareeya Chankhamjon, Yuta Tsunematsu, Mie Ishida-Ito, Yuzuka Sasa, Florian Meyer, Daniela Boettger-Schmidt, Barbara Urbansky, Klaus-Dieter Menzel, Kirstin Scherlach, Kenji Watanabe, Christian Hertweck

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   Vol. 55 ( 39 ) page: 11955 - 9   2016.9

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    The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH(2)-dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.

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  37. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids Reviewed

    Shinji Kishimoto, Michio Sato, Yuta Tsunematsu, Kenji Watanabe

    MOLECULES   Vol. 21 ( 8 )   2016.8

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    Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

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  38. New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145 Reviewed

    Hiroki Kato, Yuta Tsunematsu, Tsuyoshi Yamamoto, Takuya Namiki, Shinji Kishimoto, Hiroshi Noguchi, Kenji Watanabe

    JOURNAL OF ANTIBIOTICS   Vol. 69 ( 7 ) page: 561 - 6   2016.7

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    To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an alpha-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

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  39. Unprecedented enzyme-catalyzed generation of complexity in fumagillin-pseurotin biosynthesis

    Tsunematsu Yuta, Yamamoto Tsuyoshi, Fukutomi Manami, Kishimoto Shinji, Lin Hsiao-Ching, Tang Yi, Watanabe Kenji

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 58 ( 0 ) page: Oral34   2016

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    <p>Fumagillin and pseurotin biosynthetic gene clusters are encoded in the genome of Aspergillus fumigatus, located not side by side but together in a single region as an intermingled complex gene cluster. We have uncovered the mechanism of biosynthesis of two specific natural products, fumagillin (1) and pseurotin A (2) through mechanistic analysis of key enzymes in this study. Here we present our studies on (a) the biosynthesis of the core structure of 1, (b) the entire biosynthetic pathway of 2and (c) elucidation of the role played by PsoE (glutathione-S-transferase) and F (flavin-containing monooxygenase–methyltransferase) during the formation of the core structure of 2. </p><p>Compound 1 is a terpene–polyketide hybrid known for its antiangiogenic activity exerted through binding to human methionine aminopeptidase II. 1 has a highly oxygenated structure, and contains a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene (3). Until now, the chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted gene deletion experiments. The most significant finding from our study is that the P450 monooxygenase, Fma-P450, is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1.</p><p>Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivities. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. We systematically deleted the pseurotin biosynthetic genes in A. fumigatus and performed in vivo and in vitrocharacterizations of the tailoring enzymes to determine the biosynthetic intermediates and the gene products responsible for the formation of each intermediate. This allowed us to elucidate the main biosynthetic steps leading to the formation of 2 from the predominant precursor, azaspirene (12). The study illuminated the combinatorial nature of the pseurotin biosynthetic pathway. Most interestingly, we report the first identification of an epoxidase–methyltransferase bifunctional fusion protein PsoF that carries out C-methylation of the nascent polyketide backbone carbon atom in trans.</p><p>Lastly, we identified a novel mechanism of an enzymatic trans-to-cisisomerization of an olefin that plays a part in generating the observed chemical diversity among the pseurotin-type fungal secondary metabolites. In vitro characterizations of pseurotin biosynthetic enzymes revealed that the glutathione-S-transferase PsoE required a participation of the bifunctional epoxidase–C-methyltransferase PsoF to complete the trans-to-cis isomerization of a pathway intermediate, presynerazol (14). The PsoE–glutathione–presynerazol complex crystal structure indicated that stereospecific glutathione–presynerazol conjugate formation is the principal function of PsoE. Moreover, this study revealed PsoF to have an additional, unexpected oxidative isomerase activity, making it a trifunctional enzyme that is key to the complexity generation in pseurotin biosynthesis. </p>

    DOI: 10.24496/tennenyuki.58.0_oral34

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  40. Elucidation of Pyranonigrin Biosynthetic Pathway Reveals a Mode of Tetramic Acid, Fused gamma-Pyrone, and exo-Methylene Formation Reviewed

    Tsuyoshi Yamamoto, Yuta Tsunematsu, Hiroshi Noguchi, Kinya Hotta, Kenji Watanabe

    ORGANIC LETTERS   Vol. 17 ( 20 ) page: 4992 - 5   2015.10

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    Successful activation of the pyranonigrin biosynthetic gene cluster and gene knockout in Aspergillus niger plus in vivo and in vitro assays led to isolation of six new products, including a spiro cydobutane-containing dimeric compound, which served as the basis for the proposed comprehensive pyranonigrin biosynthetic pathway. Two redox enzymes are key to forming the characteristic fused gamma-pyrone core, and a protease homologue performs the exo-methylene formation.

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  41. Minimum Information about a Biosynthetic Gene cluster Reviewed

    Marnix H. Medema, Renzo Kottmann, Pelin Yilmaz, Matthew Cummings, John B. Biggins, Kai Blin, Irene de Bruijn, Yit Heng Chooi, Jan Claesen, R. Cameron Coates, Pablo Cruz-Morales, Srikanth Duddela, Stephanie Duesterhus, Daniel J. Edwards, David P. Fewer, Neha Garg, Christoph Geiger, Juan Pablo Gomez-Escribano, Anja Greule, Michalis Hadjithomas, Anthony S. Haines, Eric J. N. Helfrich, Matthew L. Hillwig, Keishi Ishida, Adam C. Jones, Carla S. Jones, Katrin Jungmann, Carsten Kegler, Hyun Uk Kim, Peter Koetter, Daniel Krug, Joleen Masschelein, Alexey V. Melnik, Simone M. Mantovani, Emily A. Monroe, Marcus Moore, Nathan Moss, Hans-Wilhelm Nuetzmann, Guohui Pan, Amrita Pati, Daniel Petras, F. Jerry Reen, Federico Rosconi, Zhe Rui, Zhenhua Tian, Nicholas J. Tobias, Yuta Tsunematsu, Philipp Wiemann, Elizabeth Wyckoff, Xiaohui Yan, Grace Yim, Fengan Yu, Yunchang Xie, Bertrand Aigle, Alexander K. Apel, Carl J. Balibar, Emily P. Balskus, Francisco Barona-Gomez, Andreas Bechthold, Helge B. Bode, Rainer Borriss, Sean F. Brady, Axel A. Brakhage, Patrick Caffrey, Yi-Qiang Cheng, Jon Clardy, Russell J. Cox, Rene De Mot, Stefano Donadio, Mohamed S. Donia, Wilfred A. van der Donk, Pieter C. Dorrestein, Sean Doyle, Arnold J. M. Driessen, Monika Ehling-Schulz, Karl-Dieter Entian, Michael A. Fischbach, Lena Gerwick, William H. Gerwick, Harald Gross, Bertolt Gust, Christian Hertweck, Monica Hofte, Susan E. Jensen, Jianhua Ju, Leonard Katz, Leonard Kaysser, Jonathan L. Klassen, Nancy P. Keller, Jan Kormanec, Oscar P. Kuipers, Tomohisa Kuzuyama, Nikos C. Kyrpides, Hyung-Jin Kwon, Sylvie Lautru, Rob Lavigne, Chia Y. Lee, Bai Linquan, Xinyu Liu, Wen Liu, Andriy Luzhetskyy, Taifo Mahmud, Yvonne Mast, Carmen Mendez, Mikko Metsa-Ketela, Jason Micklefield, Douglas A. Mitchell, Bradley S. Moore, Leonilde M. Moreira, Rolf Mueller, Brett A. Neilan, Markus Nett, Jens Nielsen, Fergal O'Gara, Hideaki Oikawa, Anne Osbourn, Marcia S. Osburne, Bohdan Ostash, Shelley M. Payne, Jean-Luc Pernodet, Miroslav Petricek, Joern Piel, Olivier Ploux, Jos M. Raaijmakers, Jose A. Salas, Esther K. Schmitt, Barry Scott, Ryan F. Seipke, Ben Shen, David H. Sherman, Kaarina Sivonen, Michael J. Smanski, Margherita Sosio, Evi Stegmann, Roderich D. Suessmuth, Kapil Tahlan, Christopher M. Thomas, Yi Tang, Andrew W. Truman, Muriel Viaud, Jonathan D. Walton, Christopher T. Walsh, Tilmann Weber, Gilles P. van Wezel, Barrie Wilkinson, Joanne M. Willey, Wolfgang Wohlleben, Gerard D. Wright, Nadine Ziemert, Changsheng Zhang, Sergey B. Zotchev, Rainer Breitling, Eriko Takano, Frank Oliver Gloeckner

    NATURE CHEMICAL BIOLOGY   Vol. 11 ( 9 ) page: 625 - 31   2015.9

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  42. 細胞内天然物発現法による新規遺伝子治療の開拓

    恒松 雄太

    助成研究報告集   Vol. 47   page: 4p   2015

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  43. Methylation-Dependent Acyl Transfer between Polyketide Synthase and Nonribosomal Peptide Synthetase Modules in Fungal Natural Product Biosynthesis Reviewed

    Yi Zou, Wei Xu, Yuta Tsunernatsu, Mancheng Tang, Kenji Watanabe, Yi Tang

    ORGANIC LETTERS   Vol. 16 ( 24 ) page: 6390 - 3   2014.12

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    Biochemical studies of purified and dissected fungal polyketide synthase and nonribosomal peptide synthetase (PKS-NRPS) hybrid enzymes involved in biosynthesis of pseurotin and aspyridone indicate that one a-methylation step during polyketide synthesis is a prerequisite and a key checkpoint for chain transfer between PKS and NRPS modules. In the absence of the resulting ?-methyl feature, the completed polyketide intermediate is offloaded as an a-pyrone instead of being aminoacylated by the NRPS domain. These examples illustrate that precisely timed tailoring domain activities play critical roles in the overall programming of the iterative PKS (and NRPS) functions.

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  44. Structure and Biological Activity of 8-Deoxyheronamide C from a Marine-Derived Streptomyces sp.: Heronamides Target Saturated Hydrocarbon Chains in Lipid Membranes Reviewed

    Ryosuke Sugiyama, Shinichi Nishimura, Nobuaki Matsumori, Yuta Tsunematsu, Akira Hattori, Hideaki Kakeya

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 136 ( 14 ) page: 5209 - 12   2014.4

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    Polyene macrolactams are a class of microbial metabolites, many of which show potent biological activities with unidentified modes of action. Here we report that 8-deoxyheronamide C, a new 20-membered polyene macrolactam from a marine-derived actinomycete Streptomyces sp., is a unique membrane binder. 8-Deoxyheronamide C showed a characteristic sensitivity profile against fission yeast sterol mutant cells, indicating that the metabolite targets cell membranes. We detected tight physical interaction between heronamides including 8-deoxyheronamide C and heronamide C and saturated hydrocarbon chains in lipid membranes using surface plasmon resonance experiments. We further show that heronamides induced abnormal cell wall morphology in fission yeast probably by perturbing the structure of membrane microdomains. This work will accelerate the biological and medical investigation of polyene macrolactams.

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  45. Generation of Complexity in Fungal Terpene Biosynthesis: Discovery of a Multifunctional Cytochrome P450 in the Fumagillin Pathway Reviewed

    Hsiao-Ching Lin, Yuta Tsunematsu, Sourabh Dhingra, Wei Xu, Manami Fukutomi, Yit-Heng Chooi, David E. Cane, Ana M. Calvo, Kenji Watanabe, Yi Tang

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 136 ( 11 ) page: 4426 - 36   2014.3

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    Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene beta-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.

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  46. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk in spirotryprostatins biosynthesis

    Tsunematsu Yuta, Ishikawa Noriyasu, Moriya Hisao, Noguchi Hiroshi, Watanabe Kenji

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 247   2014.3

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  47. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk in spirotryprostatins biosynthesis Reviewed

    Yuta Tsunematsu, Noriyasu Ishikawa, Hisao Moriya, Hiroshi Noguchi, Kenji Watanabe

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 247   2014.3

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    DOI: 10.1038/nchembio.1366

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  48. 細胞内天然物発現法による新規遺伝子治療の開拓

    恒松 雄太

    助成研究報告集   Vol. 46   page: 4p   2014

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  49. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk Reviewed

    Tsunematsu Yuta, Ishikawa Noriyasu, Wakana Daigo, Goda Yukihiro, Noguchi Hiroshi, Moriya Hisao, Hotta Kinya, Watanabe Kenji

    NATURE CHEMICAL BIOLOGY   Vol. 9 ( 12 ) page: 818 - +   2013.12

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    DOI: 10.1038/NCHEMBIO.1366

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  50. Targeted Disruption of Transcriptional Regulators in Chaetomium globosum Activates Biosynthetic Pathways and Reveals Transcriptional Regulator-Like Behavior of Aureonitol Reviewed

    Takehito Nakazawa, Kan'ichiro Ishiuchi, Michio Sato, Yuta Tsunematsu, Satoru Sugimoto, Yasutaka Gotanda, Hiroshi Noguchi, Kinya Hotta, Kenji Watanabe

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 135 ( 36 ) page: 13446 - 55   2013.9

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    Postgenomic analysis revealed that many microorganisms carry numerous secondary metabolite biosynthetic genes on their genome. However, activities of those putative genes are not clearly reflected in the metabolic profile of the microorganisms, especially in fungi. A recent genome mining effort is promising in discovering new natural products. However, many fungi and other organisms are not amenable to molecular genetics manipulations, making the study difficult. Here we report successful engineering of Chaetomium globosum, a known producer of various valuable natural products, that allows its genetic manipulation via targeted homologous recombination. This strain permitted us to abolish transcriptional regulators associated with epigenetic silencing of secondary metabolite biosynthetic pathways, leading to the identification of the products generated by different gene clusters and isolation of novel secondary metabolites. We were able to identify six gene clusters that are responsible for the biosynthesis of 11 natural products previously known to be produced by C. globosum, including one cytochalasan and six azaphilone-type compounds. In addition, we isolated two new compounds, mollipilin A and B, that were only recently identified in a related Chaetomium species. Furthermore, our investigation into the mechanism of biosynthesis of those natural products in C. globosum also led to the discovery of a secondary metabolite, aureonitol, that acts like a transcriptional regulator for the biosynthesis of other secondary metabolites. Similar approaches should facilitate exploration of the untapped potential of fungal biosynthetic capability and identification of various unique biological functions that those secondary metabolites possess.

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  51. Echinomycin biosynthesis Reviewed

    Michio Sato, Takehito Nakazawa, Yuta Tsunematsu, Kinya Hotta, Kenji Watanabe

    CURRENT OPINION IN CHEMICAL BIOLOGY   Vol. 17 ( 4 ) page: 537 - 45   2013.8

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    Echinomycin is an antitumor antibiotic secondary metabolite isolated from streptomycetes, whose core structure is biosynthesized by nonribosomal peptide synthetase (NAPS). The echinomycin biosynthetic pathway was successfully reconstituted in Escherichia coli. NRPS often contains a thioesterase domain at its C terminus for cyclorelease of the elongating peptide chain. Those thioesterase domains were shown to exhibit significant substrate tolerance. More recently, an oxidoreductase Ecm17, which forms the disulfide bridge in triostin A, was characterized. Surprisingly, an unrelated disulfide-forming enzyme GliT for gliotoxin biosynthesis was also able to catalyze the same reaction, providing another example of broad substrate specificity in secondary metabolite biosynthetic enzymes. Those promiscuous catalysts can be a valuable tool in generating diversity in natural products analogs we can produce heterologously.

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  52. Yeast-based genome mining, production and mechanistic studies of the biosynthesis of fungal polyketide and peptide natural products Reviewed

    Yuta Tsunematsu, Kan'ichiro Ishiuchi, Kinya Hotta, Kenji Watanabe

    NATURAL PRODUCT REPORTS   Vol. 30 ( 8 ) page: 1139 - 49   2013.8

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    In this article, we review recent successful efforts to engineer biosynthesis of several important fungal natural products through heterologous expression of relevant biosynthetic genes in Saccharomyces cerevisiae. We also describe an innovative method of rapidly cloning fungal polyketide synthase or nonribosomal peptide synthetase genes, which can be 5-20 kb or longer, from a pool of total RNA obtained from the fungus of interest using the technique we termed the "overlap extension PCR-yeast homologous recombination (ExRec)" method. The process concomitantly incorporates the cloned genes into yeast expression vectors for biosynthesis of corresponding polyketide and nonribosomal peptide compounds in our engineered S. cerevisiae strain, allowing detailed chemical characterizations to identify the activities of those previously uncharacterized biosynthetic megaenzymes. Studies reviewed here highlight yeast as a useful and versatile host not only for production of various natural products and mechanistic investigation of biosynthetic enzymes, but also for mining of uncharacterized fungal genomes for novel secondary metabolite biosynthetic pathways.

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  53. Overexpressing transcriptional regulator in fungus activates a silent biosynthetic pathway to produce novel compound

    Tsunematsu Yuta, Saruwatari Takayoshi, Sugiyama Tomohiro, Kato Hiroki, Noguchi Hiroshi, Moriya Hisao, Watanabe Kenji

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 55 ( 0 ) page: Oral35   2013

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    <p> In recent years, many secondary metabolite gene clusters, including polyketide biosynthetic genes, non-ribosomal peptide synthetase genes have been uncovered through fungal genome sequencing. However, several transcriptome analyses revealed that a lot of biosynthetic genes are not activated in conventional culture condition. If we can systematically activate such silent biosynthetic gene clusters with genetic modification, it has a possibility of discovering novel natural products with previously unidentified unique structure and biological activities. In this study, we focused putative transcription factor (TF) genes which locating in the biosynthetic gene cluster. We conducted strong activity promoter-based overexpression of TFs in Aspergillus niger, Chaetomium globosum, Arthroderma otae, Trichophyton equinum, and could successfully biosynthesize secondary metabolites 1-9. These chemical structures were characterized based on MS and NMR spectroscopic analysis. The comprehensive relationship between gene clusters and natural products has allowed us to provide insight into their biosynthetic pathway.</p>

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  54. Isolation, structure determination and biological activities of heronamides, novel membrane binders from Streptomyces sp.

    Sugiyama Ryosuke, Nishimura Shinichi, Matsumori Nobuaki, Tsunematsu Yuta, Hattori Akira, Murata Michio, Kakeya Hideaki

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 55 ( 0 ) page: Oral20   2013

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    <p>Cell membrane is one of the most challenging research subjects, partly because lipid functions at a molecular level are largely unknown. We are conducting chemical genetics approaches using compounds targeting lipids to elucidate the structure and function of cell membranes. Here we report that heronamides, polyene macrolactams from a marine-derived Streptomces sp., are a unique class of membrane binders. </p><p>We have constructed a screening system to find natural products targeting membrane lipids, using fission yeast as a model organism. During the course of the screening, we isolated heronamides, polyene macrolactams from a marine-derived Streptomyces sp. NSU893. The chemical structures of heronamides including 8-deoxyheronamide C (8-dHC, 1), a new analog, were determined by chemical conversion and spectroscopic analysis. Among heronamides, 8-dHC (1) exhibited unique cell growth inhibition pattern that was typical for membrane binders. To demonstrate the affinity between heronamides and lipid membrane, we examined surface plasmon resonance (SPR) analysis. The sensorgrams indicated that 8-dHC (1) and heronamide C (2) strongly bound lipid membranes consisting of lipids with saturated acyl/alkyl chains. In microscopic analysis, drastic morphological changes were observed when fission yeast cells were exposed to heronamides. Interestingly, this phenomenon was critically similar to that reported for a mutant of sphingolipid metabolism. These results suggest that heronamides target cell membrane probably by binding to membrane lipids, such as sphingolipids.</p><p>In conclusion, we isolated heronamides and revealed that they bound to lipid membranes consisting of saturated acyl/alkyl chains and caused a drastic morphological change in fission yeast cells. Detailed analysis of their modes of action is underway.</p>

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  55. ChemInform Abstract: Tumescenamide C, an Antimicrobial Cyclic Lipodepsipeptide from Streptomyces sp.

    Shinji Kishimoto, Yuta Tsunematsu, Shinichi Nishimura, Yutaka Hayashi, Akira Hattori, Hideaki Kakeya

    ChemInform   Vol. 43 ( 47 ) page: no - no   2012.10

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    DOI: 10.1002/chin.201247204

  56. 14 Spiro-carbon formation via common epoxidation strategy in uncommon pathway crosstalk(Oral Presentation)

    Tsunematsu Yuta, Ishikawa Noriyasu, Noguchi Hiroshi, Moriya Hisao, Watanabe Kenji

    Symposium on the Chemistry of Natural Products, symposium papers   ( 54 ) page: 79 - 84   2012.9

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    The article describes the detailed mechanism of the formation of the spiro-carbon moiety in an anti-mitotic fungal nonribosomal peptide (NRP), spirotryprostatin A, mediated by a key enzyme, FAD-dependent monooxygenase (FMO). The presence of spiro-carbon moieties is prevalent among natural products, yet the detailed mechanism of their biosynthesis is poorly characterized. Those spiro-centers remain to be a significant challenge to synthetic chemistry as well. Thus, we sought to investigate how a sterically constrained chiral spiro-carbon could be synthesized enzymatically. We focused on spirotryprostatins because of their useful biological activity and complex chemical structure involving a spiro-carbon moiety. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of complex natural product biosynthesis. What is frequently the major obstacle in conducting detailed mechanistic studies of enzymes involved in complex natural product biosynthesis is the lack of adequate supply of substrates and reference materials. This was true for studying the spiro-carbon formation in spirotryprostatins. To resolve this problem, we successfully carried out metabolic engineering of yeast for heterologous production of tryprostatins (substrates for spirotryprostatins formation) and other compounds involved in the biosynthesis of tryprostatins and spirotryprostatins using our engineered strain of Saccharomyces cerevisiae, SCKW5. Using the compounds obtained from yeast, we characterized the activity of an FMO, Afua_12060, involved in the fumiquinazoline biosynthesis and confirmed that Afua_12060 was responsible for an poxidation-driven semipinacol-type rearrangement of tryprostatin A that set up the compound for the ensuing formation of spirotryprostatin A. Comparison of our finding with the proposed biosynthetic mechanism of related fumiquinazoline and notoamide natural products revealed that FMO-catalyzed epoxidation of unactivated indole plays a key role in the formation of various complex chemical structures (namely spiro-carbon and imidazoindolone ring systems). However, what was most intriguing about our finding was the discovery of an unusual crosstalk between two biosynthetic pathways (fumiquinazoline and fumitremorgin biosynthetic pathways) that resulted in the formation of new compounds with interesting chemical features (spirotryprostatins).

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  57. Overexpressing transcriptional regulator in Chaetomium globosum activates a silent biosynthetic pathway : evaluation of shanorellin biosynthesis Reviewed

    TSUNEMATSU Yuta, ICHINOSEKI Suguru, NAKAZAWA Takehito, ISHIKAWA Noriyasu, NOGUCHI Hiroshi, HOTTA Kinya, WATANABE Kenji

      Vol. 65 ( 7 ) page: 377 - 380   2012.7

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  58. Tumescenamide C, an antimicrobial cyclic lipodepsipeptide from Streptomyces sp. Reviewed

    Shinji Kishimoto, Yuta Tsunematsu, Shinichi Nishimura, Yutaka Hayashi, Akira Hattori, Hideaki Kakeya

    TETRAHEDRON   Vol. 68 ( 27-28 ) page: 5572 - 5578   2012.7

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    Tumescenamide C, a new cyclic lipodepsipeptide, was isolated from a culture broth of an actinomycete Streptomyces sp. KUSC_F05. Tumescenamide C was a congener of tumescenamides A and B, representing a sixteen-membered ring system, consisting of two proteinogenic and three non-proteinogenic amino acids, to which a methyl-branched fatty acid was attached. The planar structure was determined by spectroscopic analysis, while its absolute stereochemistry was determined by chemical degradation and asymmetric synthesis. Tumescenamide C exhibited antimicrobial activity with high selectivity against Streptomyces species. (c) 2012 Elsevier Ltd. All rights reserved.

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  59. Overexpressing transcriptional regulator in Chaetomium globosum activates a silent biosynthetic pathway: evaluation of shanorellin biosynthesis Reviewed

    Yuta Tsunematsu, Suguru Ichinoseki, Takehito Nakazawa, Noriyasu Ishikawa, Hiroshi Noguchi, Kinya Hotta, Kenji Watanabe

    JOURNAL OF ANTIBIOTICS   Vol. 65 ( 7 ) page: 377 - 80   2012.7

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  60. Establishing a New Methodology for Genome Mining and Biosynthesis of Polyketides and Peptides through Yeast Molecular Genetics Reviewed

    Kan'ichiro Ishiuchi, Takehito Nakazawa, Takashi Ookuma, Satoru Sugimoto, Michio Sato, Yuta Tsunematsu, Noriyasu Ishikawa, Hiroshi Noguchi, Kinya Hotta, Hisao Moriya, Kenji Watanabe

    CHEMBIOCHEM   Vol. 13 ( 6 ) page: 846 - 54   2012.4

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    Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (520 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.

    DOI: 10.1002/cbic.201100798

    Web of Science

    Scopus

    PubMed

  61. 14 Spiro-carbon formation via common epoxidation strategy in uncommon pathway crosstalk(Oral Presentation)

    Tsunematsu Yuta, Ishikawa Noriyasu, Noguchi Hiroshi, Moriya Hisao, Watanabe Kenji

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 54 ( 0 ) page: 79 - 84   2012

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    Language:Japanese   Publisher:Symposium on the Chemistry of Natural Products Steering Committee  

    The article describes the detailed mechanism of the formation of the spiro-carbon moiety in an anti-mitotic fungal nonribosomal peptide (NRP), spirotryprostatin A, mediated by a key enzyme, FAD-dependent monooxygenase (FMO). The presence of spiro-carbon moieties is prevalent among natural products, yet the detailed mechanism of their biosynthesis is poorly characterized. Those spiro-centers remain to be a significant challenge to synthetic chemistry as well. Thus, we sought to investigate how a sterically constrained chiral spiro-carbon could be synthesized enzymatically. We focused on spirotryprostatins because of their useful biological activity and complex chemical structure involving a spiro-carbon moiety. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of complex natural product biosynthesis. What is frequently the major obstacle in conducting detailed mechanistic studies of enzymes involved in complex natural product biosynthesis is the lack of adequate supply of substrates and reference materials. This was true for studying the spiro-carbon formation in spirotryprostatins. To resolve this problem, we successfully carried out metabolic engineering of yeast for heterologous production of tryprostatins (substrates for spirotryprostatins formation) and other compounds involved in the biosynthesis of tryprostatins and spirotryprostatins using our engineered strain of Saccharomyces cerevisiae, SCKW5. Using the compounds obtained from yeast, we characterized the activity of an FMO, Afua_12060, involved in the fumiquinazoline biosynthesis and confirmed that Afua_12060 was responsible for an poxidation-driven semipinacol-type rearrangement of tryprostatin A that set up the compound for the ensuing formation of spirotryprostatin A. Comparison of our finding with the proposed biosynthetic mechanism of related fumiquinazoline and notoamide natural products revealed that FMO-catalyzed epoxidation of unactivated indole plays a key role in the formation of various complex chemical structures (namely spiro-carbon and imidazoindolone ring systems). However, what was most intriguing about our finding was the discovery of an unusual crosstalk between two biosynthetic pathways (fumiquinazoline and fumitremorgin biosynthetic pathways) that resulted in the formation of new compounds with interesting chemical features (spirotryprostatins).

    DOI: 10.24496/tennenyuki.54.0_79

    CiNii Research

  62. 出芽酵母発現システムを利用した天然物の生物合成 Reviewed

    恒松雄太, 守屋央朗, 渡辺賢二

    化学と生物   Vol. 50 ( 3 ) page: 163 - 174   2012

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    Language:Japanese   Publisher:Japan Society for Bioscience, Biotechnology, and Agrochemistry  

    DOI: 10.1271/kagakutoseibutsu.50.163

    CiNii Books

    CiNii Research

  63. 生合成遺伝子クラスターの高度強制発現による合成生物学が拓く有用天然物の創製 Reviewed

    中沢威人, 恒松雄太, 石川格靖, 渡辺賢二

    生物工学会誌: seibutsu-kogaku kaishi   Vol. 90 ( 6 ) page: 289 - 292   2012

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    Language:Japanese  

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  64. P-43 Pentadepsin A, a Novel Lipodepsipeptide Produced by Streptomyces sp.(Poster Presentation)

    Kishimoto Shinji, Tsunematsu Yuta, Nishimura Shinichi, Hattori Akira, Kakeya Hideaki

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 53 ( 0 ) page: 397 - 402   2011

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    Microbes are a rich source of bioactive metabolites; they represent a wide variety of chemical structures and specific biological activities. In our continuing search for bioactive metabolites from microbes, a culture broth of an actinomycete strain of the genus Streptomyces, from which several bioactive secondary metabolites were isolated, was found to contain a novel antimicrobial lipodepsipeptide, designated pentadepsin A (1). Herein, we report the isolation, structure elucidation and biological activity of pentadepsin A. The molecular formula of 1 was established as C37H57N508 on the basis of HR-FABMS and NMR spectral data. The planar structure of 1 was elucidated by 2D NMR analysis; it represented a sixteen-membered ring system, consisting of five amino acids, to which a unique acyl group 2,4-dimethyl heptanoic acid (Dmh) was linked through an amide bond. The absolute stereochemistry of amino acid residues was determined by advanced Marfey's method, while that of Dmh residue was partially elucidated by PGME method. For the full elucidation of the side chain structure, (2S,4R)-Dmh and (2S,4S)-Dmh were synthesized via Evans alkylation. Spectroscopic comparison of Dmh obtained from 1 with the synthesized ones revealed that the side chain of 1 is (2S,4S)-2,4-dimethylheptanoic acid. Pentadepsin A selectively inhibited cell growth of Streptomyces species, while exhibited no growth inhibition against other bacteria tested and no cytotoxic effect mammalian cells. Detailed analysis of the mechanism of action of pentadepsin A is underway.

    DOI: 10.24496/tennenyuki.53.0_397

    CiNii Books

    CiNii Research

  65. VCR/AraC chemotherapy and ND-CNS-LCH.

    Imashuku S, Shioda Y, Tsunematsu Y, Imamura T, Morimoto A, Japan LCH Study Group

    Pediatric blood & cancer   Vol. 55 ( 1 ) page: 215 - 6   2010.7

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    Language:English   Publisher:Pediatric Blood and Cancer  

    DOI: 10.1002/pbc.22478

    Web of Science

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    PubMed

  66. Isolation, Structure Elucidation and Synthesis of Tryptopeptin A,a Novel TGF-β Signaling Pathway Inhibitor Produced by Streptomyces sp.

    TSUNEMATSU Yuta, OISHI Shinya, FUJII Nobutaka, NISHIMURA Shinichi, HATTORI Akira, KAKEYA Hideaki

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 52 ( 0 ) page: 397 - 402   2010

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    Language:Japanese   Publisher:Symposium on the Chemistry of Natural Products Steering Committee  

    Transforming growth factor-β (TGF-β) facilitates tumor growith and metastasis in advanced cancer. Discovery of novel TGF-β signaling inhibitors may open a new strategy for treatment of cancer patients. However, few clinically-promising drugs that block the TGF-β signaling pathway have been reported. Therefore, we are investigating novel TGF-β signaling inhibitors from natural sources, including microbial metabolites. For the screening assay, we established a luciferase reporter cell line from mink lung epithelial cells, Mv1Lu, to monitor the TGF-β-induced signal. Among over 2000 microbial extracts screened, an extract from an actinomycete Streptomyces sp. was found to inhibit the luciferase activity without cytotoxicity. Three litters of the culture broth were extracted with n-BuOH, filtered and concentrated. An active substance, named tryptopeptin A, was obtained by using silica gel chromatography followed by reverse-phase HPLC, as a colorless solid (3.6mg, 0.023%). Detailed MS and NME spectroscopic analyses revealed that tryptopeptin A was a novel acylated tripeptede, somprising a isovaleric acid, an N-methylvaline, a threonine and a C-terminally modified tryptophan in which carboxylic acid was replaced with an α,β-epoxyketone gropu. The absolute stereochemistries of tryptopeptin A were determined by Marfey's method and a synthetic approach: the physicochemical properties of the synthesized compound were idintical with those of the natural product. Tryptopeptin A potently inhibited TGF-β-dependent luciferase activity without cytotoxicity, suggesting the presence of specific cellular target molecule(s). Structure-activity relationships and modes of action of tryptopeptin A will also be discussed.

    DOI: 10.24496/tennenyuki.52.0_397

    CiNii Research

  67. LANGERHANS CELL HISTIOCYTOSIS INVOLVING THE THYROID ASSOCIATED WITH CERVICAL SOFT TISSUE MASS IN CHILDREN: REPORT OF THREE CASES

    Shioda Yoko, Morimoto Akira, Ueyama Junichi, Shimada Hiroyuki, Fujimoto Junichiro, Tsunematsu Yukiko, Imashuku Shinsaku

    PEDIATRIC BLOOD & CANCER   Vol. 53 ( 4 ) page: 690 - 690   2009.10

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  68. Symbiospirols: Novel Long Carbon-Chain Compounds Isolated from Symbiotic Marine Dinoflagellate Symbiodinium sp. Reviewed

    Yuta Tsunematsu, Osamu Ohno, Kaori Konishi, Kaoru Yamada, Masami Suganuma, Daisuke Uemura

    ORGANIC LETTERS   Vol. 11 ( 10 ) page: 2153 - 6   2009.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER CHEMICAL SOC  

    Symbiospirols A, B, and C, long carbon-chain compounds with a molecular weight of 1207 were isolated from the cultured symbiotic dinoflagellate Symbiodinium sp. Their planar structures and partial relative stereochemistries were elucidated based on 1D and 2D NMR spectra and a degradation reaction. Symbiospirols consisted of a C67-linear chain with a beta,beta&apos;-dihydroxyl ketone moiety, eight hydroxyl groups, one tetrahydropyran ring, and two 1,6-dioxaspiro[4,4]nonane rings. Symbiospirol A had an inhibitory effect against L-phosphatidylserine-induced PKC activation.

    DOI: 10.1021/ol900299x

    Web of Science

    Scopus

    PubMed

  69. 2 Isolation and structure determination of bioactive polyols using a novel degradation reaction(Oral Presentation)

    Han Chunguang, Hanif Novriyandi, Tsunematsu Yuta, Ohno Osamu, Yamano Yoshi, Kitamura Makoto, Yamada Kaoru, Kita Masaki, Suganuma Masami, Uemura Daisuke

    Symposium on the Chemistry of Natural Products, symposium papers   ( 50 ) page: 35 - 40   2008.9

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    Language:Japanese   Publisher:Symposium on the chemistry of natural products  

    Dinoflagellates are widely known as the rich source of biologically active and structural unique secondary metabolites. Among them, large polyols and polyethers composed of a long carbon backbone that is highly functionalized by oxygen, as we say "super-carbon-chain compounds (SCCs)", are some of the most unusual compounds. In our quest for SCCs from marine dinoflagellates, we isolated a novel spiroketal compound, symbiospirol A, from Symbiodinium sp. that was the same strain which produced symbiodinolide. Its planar structure and partial relative stereochemistries were elucidated based on 1D and 2D-NMR and degradation reaction. Symbiospirol A was shown to inhibit PKC activation induced by phosphatidylserine. Symbiodinolide is the typical SCC with a molecular weight of 2,860, which we isolated from the dinoflagellate Symbiodinium sp. The chemical degradation reaction using the 2nd-generation Hoveyda-Grubbs catalyst was employed for the stereochemical assignment at 62-membered macrolactone of symbiodinolide. The absolute configurations at several chiral centers were determined by spectroscopic analysis of MTPA diesters. Further chemical degradation studies using the 2nd-generation Grubbs' catalyst have performed new reaction. In addition, the complete stereochemical assignment of symbiodinolide is in progress. In our continuing search for novel bioactive metabolites from symbiotic marine dinoflagellates, we found that the water layer from the symbiotic dinoflagellate with jellyfish Mastigias papua displayed considerable activity against the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC). Further purification of the water layer guided by the bioassay and mass measurement led to the isolation of an unknown bioactive SCC, which we named symbiopolyol, possessing the molecular weight of 1,220. Studies on the isolation, structure elucidation, and biological activities of symbiopolyol will be described.

    DOI: 10.24496/tennenyuki.50.0_35

    CiNii Books

  70. Structure of zamamistatin - a correction Reviewed

    Masaki Kita, Yuta Tsunematsu, Ichiro Hayakawa, Hideo Kigoshi

    TETRAHEDRON LETTERS   Vol. 49 ( 37 ) page: 5383 - 5384   2008.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PERGAMON-ELSEVIER SCIENCE LTD  

    The structure of zamamistatin, an antibacterial dibromotyrosine derivative, has been revised. Its structure was not exo- or endo-type dimer 1 or 2 which were described previously, but was identical to a dibromophenylpyruvic acid derivative, aeroplysinin-1 (3). (c) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.tetlet.2008.06.125

    Web of Science

    Scopus

  71. 2 Isolation and structure determination of bioactive polyols using a novel degradation reaction(Oral Presentation)

    Han Chunguang, Hanif Novriyandi, Tsunematsu Yuta, Ohno Osamu, Yamano Yoshi, Kitamura Makoto, Yamada Kaoru, Kita Masaki, Suganuma Masami, Uemura Daisuke

    Symposium on the Chemistry of Natural Products, symposium papers   Vol. 50 ( 0 ) page: 35 - 40   2008

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    Language:Japanese   Publisher:Symposium on the Chemistry of Natural Products Steering Committee  

    Dinoflagellates are widely known as the rich source of biologically active and structural unique secondary metabolites. Among them, large polyols and polyethers composed of a long carbon backbone that is highly functionalized by oxygen, as we say "super-carbon-chain compounds (SCCs)", are some of the most unusual compounds. In our quest for SCCs from marine dinoflagellates, we isolated a novel spiroketal compound, symbiospirol A, from Symbiodinium sp. that was the same strain which produced symbiodinolide. Its planar structure and partial relative stereochemistries were elucidated based on 1D and 2D-NMR and degradation reaction. Symbiospirol A was shown to inhibit PKC activation induced by phosphatidylserine. Symbiodinolide is the typical SCC with a molecular weight of 2,860, which we isolated from the dinoflagellate Symbiodinium sp. The chemical degradation reaction using the 2nd-generation Hoveyda-Grubbs catalyst was employed for the stereochemical assignment at 62-membered macrolactone of symbiodinolide. The absolute configurations at several chiral centers were determined by spectroscopic analysis of MTPA diesters. Further chemical degradation studies using the 2nd-generation Grubbs' catalyst have performed new reaction. In addition, the complete stereochemical assignment of symbiodinolide is in progress. In our continuing search for novel bioactive metabolites from symbiotic marine dinoflagellates, we found that the water layer from the symbiotic dinoflagellate with jellyfish Mastigias papua displayed considerable activity against the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC). Further purification of the water layer guided by the bioassay and mass measurement led to the isolation of an unknown bioactive SCC, which we named symbiopolyol, possessing the molecular weight of 1,220. Studies on the isolation, structure elucidation, and biological activities of symbiopolyol will be described.

    DOI: 10.24496/tennenyuki.50.0_35

    CiNii Research

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Books 6

  1. Dissecting Biosynthesis of Natural Products Toward Drug Discovery Reviewed

    Yuta Tsunematsu( Role: Sole author)

    New Tide of Natural Product Chemistry  2023.6 

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  2. 担子菌休眠型生合成遺伝子の覚醒による 新規天然物の発見とその菌糸成長・子実体形成作用

    恒松雄太( Role: Sole author)

    アグリバイオ11月号 北隆館  2021.10 

  3. 21世紀における構造未決定天然物 大腸発がんリスク因子コリバクチンの生合成

    恒松 雄太

    有機合成化学協会誌, 76, 490-493, 2018.  2018.5 

  4. 新規疾患治療法開発のヒントは「微生物の生存戦略」に隠されている?

    恒松 雄太( Role: Sole author ,  54, 458, 2018)

    ファルマシア, 54, 458, 2018.  2018.5 

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    Language:Japanese

  5. 生合成遺伝子クラスターの高度強制発現による合成生物学が拓く有用天然物の創製

    中沢威人, 恒松雄太, 石川格靖, 渡辺賢二

    生物工学会誌 90, 289-292, 2012. 

  6. 出芽酵母発現システムを利用した天然物の生物合成

    恒松雄太, 守屋央朗, 渡辺賢二

    化学と生物 50, 163-174, 2012. 

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MISC 14

  1. コリバクチン産生大腸菌のin vitro遺伝毒性試験

    久冨優太, 小田美光, 下原千晶, 恒松雄太, 佐藤道大, 平山裕一郎, 三好規之, 岩下雄二, 吉川悠子, 八木孝司, 椙村春彦, 若林敬二, 渡辺賢二, 川西優喜

    日本環境変異原学会大会プログラム・要旨集   Vol. 47th   page: 105   2018.10

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    Language:Japanese  

    J-GLOBAL

  2. 検出困難な遺伝毒性物質コリバクチンを見出すためのDNA付加体の解析

    平山裕一郎, 田島悠也, 恒松雄太, 三好規之, 若林敬二, 渡辺賢二

    日本生薬学会年会講演要旨集   Vol. 65th   page: 67 - 67   2018.8

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    J-GLOBAL

  3. 大腸がん患者から分離された遺伝毒性物質コリバクチン陽性菌の解析

    吉川 悠子, 恒松 雄太, 松崎 信生, 平山 裕一郎, 佐藤 道大, 三好 規之, 岩下 雄二, 椙村 春彦, 若林 敬二, 渡辺 賢二

    日本細菌学雑誌   Vol. 73 ( 1 ) page: 56 - 56   2018.2

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    Language:Japanese   Publisher:日本細菌学会  

  4. 大腸がん患者から分離された遺伝毒性物質コリバクチン陽性菌の解析

    吉川悠子, 吉川悠子, 恒松雄太, 松崎信生, 平山裕一郎, 佐藤道大, 三好規之, 岩下雄二, 椙村春彦, 若林敬二, 渡辺賢二

    日本細菌学雑誌(Web)   Vol. 73 ( 1 ) page: 56(J‐STAGE) - 56   2018.2

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    J-GLOBAL

  5. 大腸がん患者から分離された遺伝毒性物質コリバクチン陽性菌の解析

    吉川 悠子, 恒松 雄太, 松崎 信生, 平山 裕一郎, 佐藤 道大, 三好 規之, 岩下 雄二, 椙村 春彦, 若林 敬二, 渡辺 賢二

    日本細菌学雑誌   Vol. 73 ( 1 ) page: 56 - 56   2018.2

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  6. 大腸がんリスク因子物質コリバクチンの生産菌検出に関する化学的研究

    平山裕一郎, 松崎信生, 玉舟亮太, 恒松雄太, 佐藤道大, 吉川悠子, 吉川悠子, 三好規之, 岩下雄二, 椙村春彦, 若林敬二, 武藤倫弘, 石川秀樹, 渡辺賢二

    がん予防学術大会プログラム・抄録集   Vol. 2018   page: 51   2018

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    J-GLOBAL

  7. 腸内細菌叢を起源とする遺伝毒性物質コリバクチンの科学分析手法の確立と日本人コホートにおける発がんとの関連性の解析

    恒松雄太, 平山裕一郎, 桝谷貴洋, 松崎信生, 佐藤道大, 吉川悠子, 三好規之, 若林敬二, 武藤倫弘, 村上晴香, 宮地元彦, 石川秀樹, 渡辺賢二

    がん予防学術大会プログラム・抄録集   Vol. 2017   page: 29 - 3001   2017.9

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    Language:Japanese   Publisher:日本癌学会  

    J-GLOBAL

  8. 微量天然物spirotryprostatin類生物全合成によるスピロ環形成機構の解明

    恒松雄太, 石川格靖, 若菜大悟, 合田幸広, 野口博司, 守屋央朗, 堀田欣也, 渡辺賢二

    日本生薬学会年会講演要旨集   Vol. 60th   page: 101   2013.8

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    J-GLOBAL

  9. 新規ポリエンマクロラクタム8-deoxyheronamide Cの単離・構造決定とその生物活性

    杉山龍介, 西村慎一, 恒松雄太, 服部明, 掛谷秀昭

    日本薬学会年会要旨集(CD-ROM)   Vol. 133rd   2013

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  10. 海洋由来放線菌が産生するheronamide類の単離と生物活性

    杉山龍介, 西村慎一, 松森信明, 恒松雄太, 服部明, 村田道雄, 掛谷秀昭

    天然有機化合物討論会講演要旨集(Web)   Vol. 55th   2013

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  11. 新規TGF‐βシグナル伝達阻害剤tryptopeptin Aおよび分子プローブの合成研究

    KASAI SHOTA, SAKAI YUKI, TSUNEMATSU YUTA, HATTORI AKIRA, OISHI SHIN'YA, FUJII NOBUTAKA, NISHIMURA SHIN'ICHI, KAKEYA HIDEAKI

    日本薬学会年会要旨集   Vol. 132nd ( 2 ) page: 221   2012.3

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    J-GLOBAL

  12. 放線菌の生産する新規抗がんリード化合物tryptopeptin Aの化学構造決定

    TSUNEMATSU YUTA, HATTORI AKIRA, OISHI SHIN'YA, FUJII NOBUTAKA, NISHIMURA SHIN'ICHI, KAKEYA HIDEAKI

    日本薬学会年会要旨集   Vol. 131st ( 2 ) page: 104   2011.3

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    J-GLOBAL

  13. 放線菌が生産する新規TGF‐βシグナル伝達阻害剤tryptopeptin Aの単離・構造解析と合成

    TSUNEMATSU YUTA, OISHI SHIN'YA, FUJII NOBUTAKA, NISHIMURA SHIN'ICHI, HATTORI AKIRA, KAKEYA HIDEAKI

    天然有機化合物討論会講演要旨集   Vol. 52nd ( 52 ) page: 397 - 402   2010.9

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    Language:Japanese   Publisher:Symposium on the chemistry of natural products  

    Transforming growth factor-β (TGF-β) facilitates tumor growith and metastasis in advanced cancer. Discovery of novel TGF-β signaling inhibitors may open a new strategy for treatment of cancer patients. However, few clinically-promising drugs that block the TGF-β signaling pathway have been reported. Therefore, we are investigating novel TGF-β signaling inhibitors from natural sources, including microbial metabolites. For the screening assay, we established a luciferase reporter cell line from mink lung epithelial cells, Mv1Lu, to monitor the TGF-β-induced signal. Among over 2000 microbial extracts screened, an extract from an actinomycete Streptomyces sp. was found to inhibit the luciferase activity without cytotoxicity. Three litters of the culture broth were extracted with n-BuOH, filtered and concentrated. An active substance, named tryptopeptin A, was obtained by using silica gel chromatography followed by reverse-phase HPLC, as a colorless solid (3.6mg, 0.023%). Detailed MS and NME spectroscopic analyses revealed that tryptopeptin A was a novel acylated tripeptede, somprising a isovaleric acid, an N-methylvaline, a threonine and a C-terminally modified tryptophan in which carboxylic acid was replaced with an α,β-epoxyketone gropu. The absolute stereochemistries of tryptopeptin A were determined by Marfey's method and a synthetic approach: the physicochemical properties of the synthesized compound were idintical with those of the natural product. Tryptopeptin A potently inhibited TGF-β-dependent luciferase activity without cytotoxicity, suggesting the presence of specific cellular target molecule(s). Structure-activity relationships and modes of action of tryptopeptin A will also be discussed.

    DOI: 10.24496/tennenyuki.52.0_397

    CiNii Books

    J-GLOBAL

  14. HIF‐1機能制御化合物の評価系構築と探索研究

    HATTORI AKIRA, TSUNEMATSU YUTA, OTSUKA SAORI, TAKASU YASUAKI, NISHIMURA SHIN'ICHI, YAKURA TOORU, OISHI SHIN'YA, FUJII NOBUTAKA, SHIMIZU SHIRO, OSADA HIROYUKI, KAKEYA HIDEAKI

    日本薬学会年会要旨集   Vol. 129th ( 2 ) page: 183   2009.3

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    J-GLOBAL

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Presentations 56

  1. 恒松雄太, 渡辺賢二:Harnessing the Biosynthetic Pathway in Filamentous Fungus, Osaka University-Universiti Brunei Darussalam Joint symposium, 2018年2月21日 (Brunei Darussalam)2018年2月21日(ポスター)

  2. (受賞講演)恒松雄太:糸状菌ゲノム情報を活用した天然物探索とその生合成機構解明, 日本生薬学会第66回年会(東京), 2019年9月22-23日 (東京都港区)(口頭)

  3. (招待講演)恒松雄太, 渡辺賢二:Dissecting and harnessing fungal biosynthetic pathway, International Congress on Pure & Applied Chemistry Langkawi (ICPAC Langkawi) 2018, 2018年10月30日-11月2日 (Langkawi, Malaysia)2018年10月30日-11月2日(口頭)

  4. (招待講演)恒松雄太:生合成遺伝子制御に基づく天然物創薬リバイバル, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  5. (招待講演)恒松雄太:生合成遺伝子制御に基づく天然物創薬リバイバル, 第 7 回慶應有機化学若手シンホ_シ_ウム, 2019年5月11日 (神奈川県横浜市)(口頭)

  6. (招待講演)恒松雄太:糸状菌ゲノムが拡張する天然有機化合物研究, 糸状菌分子生物学研究会若手の会第7回ワークショップ, 2019年11月5-6日 (北海道空知郡)(口頭)

  7. (招待講演)恒松雄太:糸状菌ゲノム情報を活用した新規天然物の探索とその生合成, 静岡大学超領域研究推進本部 第12回超領域研究会, 2018年12月3日 (静岡県静岡市)2018年12月3日(口頭)

  8. 高西潤, 桝谷貴洋, 中沢威人, 恒松雄太, 佐藤道大, 渡辺賢二:レギュレータ-遺伝子改変法によるCoprinopsis属担子菌の新規天然物生合成経路活性化, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  9. 籠浦倫美, 佐藤道大, 恒松雄太, 竹仲由希子, 棚橋孝雄, 渡辺賢二:共生型子嚢菌Pyrenula sp.に由来するPyrenulic acid類の生合成遺伝子の同定, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  10. 籠浦倫美, 佐藤道大, 恒松雄太, 竹仲由希子, 棚橋孝雄, 渡辺賢二:共生型子嚢菌Pyrenula sp.に由来するPyrenulic acid類の生合成遺伝子の同定, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  11. 鈴木七海, 恒松雄太, 平山裕一郎, 尾形勇二, 佐藤道大, 辻本恭, 内山奈穂子, 袴塚高志, 合田幸広, 渡辺賢二:大腸がんリスク因子物質コリバクチン関連化合物の構造決定, 日本生薬学会第66回年会(東京), 2019年9月22-23日 (東京都港区)(口頭)

  12. 高西潤, 桝谷貴洋, 中沢威人, 恒松雄太, 佐藤道大, 渡辺賢二:Coprinopsis属担子菌のレギュレータ−遺伝子操作による化合物生産, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  13. 高西潤, 桝谷貴洋, 中沢威人, 恒松雄太, 佐藤道大, 渡辺賢二:キノコ由来二次代謝産物の過剰生産系の構築, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  14. 高西潤, 桝谷貴洋, 中沢威人, 恒松雄太, 佐藤道大, 渡辺賢二:レギュレータ-遺伝子改変法によるCoprinopsis属担子菌の新規天然物生合成経路活性化, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  15. 田村優依, 桝谷貴洋, 恒松雄太, 佐藤道大, 渡辺賢二:レギュレーターを利用した冬虫夏草代謝産物の生産変化, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  16. "前田直哉, 恒松雄太, 原幸大, 橋本博, 渡辺賢二:Aspirochlorine生合成経路におけるスヒ_ロ環形成を担う新規酵素の発見と機能 解析, 第61回天然有機化合物討論会, 2019年9月11-13日 (広島県広島市)(ポスター)"

  17. (招待講演)渡辺賢二, 恒松雄太, 平山裕一郎, 東口ふみ, 佐藤道大, 吉川悠子, 三好規之, 岩下雄二, 椙村春彦, 武藤倫弘, 石川秀樹, 若林敬二:大腸か_んリスク因子コリハ_クチン産生菌の増殖抑制を目的としたハ_イロッ ト介入試験, がん予防学術大会2019札幌, 2019年6月28-29日 (北海道札幌市)(口頭)

  18. 久冨優太, 小田美光, 下原千晶, 恒松雄太, 佐藤道大, 平山裕一郎, 三好規之, 岩下雄二, 吉川悠子, 八木孝司, 椙村春彦, 若林敬二, 渡辺賢二, 川西優喜:コリバクチン産生大腸菌のin vitro遺伝毒性試験, 日本環境変異原学会第47回大会, 2018年11月2-3日 (京都府京都市)2018年11月2-3日(ポスター)

  19. 佐藤道大, 平山裕一郎, 恒松雄太, 東口ふみ, 吉川悠子, 三好規之, 岩下雄二, 椙村春彦, 若林敬二, 武藤倫弘, 石川秀樹, 渡辺賢二:遺伝毒性物質コリハ_クチン生産菌の高感度多検体検出法の確立, がん予防学術大会2019札幌, 2019年6月28-29日 (北海道札幌市)(口頭)

  20. 佐藤道大, 籠浦倫美, 恒松雄太, 竹仲由希子, 棚橋孝雄, 渡辺賢二:共生型子嚢菌Pyrenula sp.由来pyrenulic acid類の生合成研究, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  21. 佐藤道大, 籠浦倫美, 恒松雄太, 竹仲由希子, 棚橋孝雄, 渡辺賢二:共生型子嚢菌Pyrenula sp.由来pyrenulic acid類の生合成研究, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  22. 前田直哉, 恒松雄太, Hertweck Christian, 渡辺賢二:Aspirochlorine 生合成経路におけるスピロ環形成を担う新規酵素の発見と機能解析, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  23. 前田直哉, 恒松雄太, Hertweck Christian, 渡辺賢二:Aspirochlorine 生合成経路におけるスピロ環形成を担う新規酵素の発見と機能解析, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  24. 前田直哉, 恒松雄太, 原幸大, 橋本博, Christian Hertweck, 渡辺賢二:Aspirochlorine 生合成経路におけるスピロ環形成酵素の発見と機能解析, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  25. 前田直哉, 恒松雄太, 渡辺賢二:Revealing an unprecedented biosynthetic pathway in Aspergillus oryzae, 30th International Symposium on the Chemistry of Natural Products and the 10th International Congress on Biodiversity (ISCNP30 & ICOB10), 2018年11月25-29日 (Athens, Greec

  26. 岸本真治, 原幸大, 石川格靖, 山田陽香, 恒松雄太, 橋本博, Kendall N. Houk, Yi Tang, 渡辺賢二:キノロン骨格を形成する糸状菌酵素シクロペナーゼの反応機構解析, 第60回天然有機化合物討論会, 2018年9月26-28日 (福岡県久留米市)2018年9月26-28日(口頭)

  27. 岸本真治, 松下拓磨, 原幸大, 橋本博, 恒松雄太, 渡辺賢二:ポリケチド鎖をメチル化するPsoF-MTの構造解析とメチル化部位選択性の解明, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  28. 岸本真治, 横山葵, 佐藤道大, 前田直哉, 恒松雄太, 原幸大, 橋本博, 渡辺賢二:立体選択的なDiels-Alder反応を触媒する酵素CghAの触媒機構解明, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  29. 岸本真治, 横山葵, 佐藤道大, 前田直哉, 恒松雄太, 原幸大, 橋本博, 渡辺賢二:糸状菌由来酵素 CghA による立体選択的な Diels-Alder 反応の機構解明, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(ポスター)

  30. 岸本真治, 横山葵, 佐藤道大, 前田直哉, 恒松雄太, 原幸大, 橋本博, 渡辺賢二:糸状菌由来酵素 CghA による立体選択的な Diels-Alder 反応の機構解明, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  31. 岸本真治, 横山葵, 成田一仁, 佐藤道大, 前田直哉, 恒松雄太, 原幸大, 橋本博, 渡辺賢二:糸状菌由来Diels_Alderase CghAの反応機構解明, 日本生薬学会第66回年会(東京), 2019年9月22-23日 (東京都港区)(口頭)

  32. 平山裕一郎, 松崎信生, 玉舟亮太, 恒松雄太, 佐藤道大, 吉川悠子, 三好規之, 岩下雄二, 椙村春彦, 若林敬二, 武藤倫弘, 石川秀樹, 渡辺賢二:大腸がんリスク因子物質コリバクチンの生産菌検出に関する化学的研究, がん予防学術大会2018高松, 2018年6月27-28日 (香川県高松市)2018年6月27-28日(ポスター)

  33. 平山裕一郎, 玉舟亮太, 恒松雄太, 渡辺賢二:大腸がんリスク因子物質コリバクチン関連化合物の特定, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  34. 平山裕一郎, 玉舟亮太, 恒松雄太, 渡辺賢二:大腸がんリスク因子物質コリバクチン関連化合物の特定, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  35. 平山裕一郎, 玉舟亮太, 松崎信生, 恒松雄太, 渡辺賢二:大腸がんリスク因子物質コリバクチンの生産菌検出に関する化学的研究, 第22回天然薬物の開発と応用シンポジウム, 2018年10月7-8日 (熊本県熊本市)2018年10月7-8日(ポスター)

  36. 平山裕一郎, 田島悠也, 恒松雄太, 三好規之, 若林敬二, 渡辺 賢二:検出困難な遺伝毒性物質コリバクチンを見出すためのDNA付加体の解析, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  37. 平山裕一郎, 鈴木七海, 佐藤道大, 恒松雄太, 内山奈穂子, 合田幸広, 渡辺賢二:大腸菌が生合成する大腸がんリスク因子化合物コリバクチンの検出と構造解明, 第61回天然有機化合物討論会, 2019年9月11-13日 (広島県広島市)(ポスター)

  38. 恒松雄太, 吉川悠子, 三好規之, 武藤倫弘, 山地太樹, 張萌琳, 岩崎基, 松田尚久, 細見晃司, 國澤純, 若林敬二, 石川秀樹, 渡辺賢二:大腸発がんリスク因子コリバクチンの生産性を制御する要因の解明, がん予防学術大会2019札幌, 2019年6月28-29日 (北海道札幌市)(ポスター)

  39. 恒松雄太, 渡辺賢二:Dissecting and harnessing fungal biosynthetic pathway, 30th International Symposium on the Chemistry of Natural Products and the 10th International Congress on Biodiversity (ISCNP30 & ICOB10), 2018年11月25-29日 (Athens, Greece)2018年11月25-29日(口頭)

  40. 恒松雄太:ヒト細菌叢を構成する微生物由来の天然物化学, 第54回天然物談話会, 2019年7月3-5日 (北海道札幌市)(ポスター)

  41. 成田一仁, 岸本真治, 横山葵, 恒松雄太, 佐藤道大, 渡辺賢二:Diels-Alder反応を触媒する酵素CghAの基質合成および反応解析, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  42. 成田一仁, 岸本真治, 横山葵, 恒松雄太, 佐藤道大, 渡辺賢二:Diels-Alder反応を触媒する酵素CghAの基質合成および反応解析, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  43. 成田一仁, 岸本真治, 横山葵, 恒松雄太, 佐藤道大, 渡辺賢二:立体選択的反応機構の解明を目指したDiels-Alder反応を触媒するCghAの基質合成研究, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  44. 成田一仁, 岸本真治, 横山葵, 恒松雄太, 佐藤道大, 渡辺賢二:立体選択的反応機構の解明を目指したDiels-Alder反応を触媒するCghAの基質合成研究, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  45. 松下拓磨, 原幸大, 恒松雄太, 橋本博, 渡辺賢二:フラビン依存型モノオキシゲナーゼによるスピロ環化形成機構の解明, 日本生薬学会第66回年会(東京), 2019年9月22-23日 (東京都港区)(口頭)

  46. 松下拓磨, 岸本真治, 山本剛, 原幸大, 恒松雄太, 橋本博, 渡辺賢二:PsoFのメチルトランスフェラーセ_ト_メインの構造解析, 2017年度量子ビームサイエンスフェスタ, 2018年3月2-4日 (茨城県つくば市)2018年3月2-4日(ポスター)

  47. 松下拓磨, 岸本真治, 山本剛, 原幸大, 恒松雄太, 橋本博, 渡辺賢二:PsoFメチルトランスフェラーゼドメインの基質認識機構の解明と酵素機能の改変, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  48. 桝谷貴洋, 平山裕一郎, 高西潤, 恒松雄太, 佐藤道大, 大高潤之介, 本山高幸, 長田裕之, 渡辺賢二:複雑な酸化経路を含むキノコ由来lagopodin類の生合成研究, 日本薬学会第139年会(千葉), 2019年3月20-23日 (千葉県千葉市)(口頭)

  49. 桝谷貴洋, 平山裕一郎, 高西潤, 恒松雄太, 佐藤道大, 大高潤之介, 本山高幸, 長田裕之, 渡辺賢二:複雑な酸化経路を含むキノコ由来lagopodin類の生合成研究, 日本農芸化学会2019年度東京大会, 2019年3月24-27日 (東京都世田谷区)(口頭)

  50. 桝谷貴洋, 平山裕一郎, 高西潤, 田村優依, 中沢威人, 恒松雄太, 佐藤道大, 渡辺賢二:キノコ由来天然物ラゴポジン類の生合成研究, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  51. 横山葵, 佐藤 道大, 前田 直哉, 恒松 雄太, 原 幸大, 橋本 博, 渡辺 賢二:酵素_生成物複合体を用いたCghAの立体選択的反応機構の解明, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  52. 横山葵, 佐藤道大, 前田直哉, 恒松雄太, 原幸大, 橋本博, 渡辺賢二:酵素_生成物複合体を用いたCghAの立体選択的反応機構の解明, 日本生薬学会第65回年会(広島), 2018年9月16-17日 (広島県広島市)2018年9月16-17日(口頭)

  53. 横山葵, 前田直哉, 岸本真治, 佐藤道大, 原幸大, 恒松雄太, 橋本博, 渡辺賢二:新規構造を持つ環化酵素CghAのX線結晶構造解析とその立体選択的反応機構 , 2017年度量子ビームサイエンスフェスタ, 2018年3月2-4日 (茨城県つくば市)2018年3月2-4日(ポスター)

  54. 浅井しほり, 恒松雄太, 高西潤, 桝谷貴洋, 佐藤道大, 大高潤之介, 本山高幸, 長田裕之, 渡辺賢二:キノコ由来天然物lagopodin類の生合成研究, 日本生薬学会第66回年会(東京), 2019年9月22-23日 (東京都港区)(口頭)

  55. 玉舟亮太, 平山裕一郎, 恒松雄太, 佐藤道大, 渡辺賢二:大腸がんリスク因子物質コリバクチンの生産菌を検出する分子プローブ, 日本病院薬剤師会東海ブロック・日本薬学会東海支部合同学術大会2018, 2018年11月14日 (静岡県静岡市)2018年11月14日(口頭)

  56. 玉舟亮太, 平山裕一郎, 恒松雄太, 渡辺賢二:大腸がんリスク因子物質コリバクチンの生産菌を検出する分子プローブ, 第22回天然薬物の開発と応用シンポジウム, 2018年10月7-8日 (熊本県熊本市)2018年10月7-8日(口頭)

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KAKENHI (Grants-in-Aid for Scientific Research) 6

  1. 生物圏と物質の統合的情報解析によるホロビオント分子科学の創成 International coauthorship

    Grant number:23H03820  2023.4 - 2026.3

    学術変革領域研究(B)

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\20280000 ( Direct Cost: \15600000 、 Indirect Cost:\4680000 )

  2. 共生体が持つ物質の多様性と生態系における新機能の解明 計画研究 International coauthorship

    Grant number:23H03823  2023.4 - 2026.3

    学術変革領域研究(B)

    恒松雄太

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\50440000 ( Direct Cost: \38800000 、 Indirect Cost:\11640000 )

  3. The molecular and functional generality of the Basidiomycetous siderophore that promotes mycelial growth and fruiting body formation

    Grant number:20K05866  2020.4 - 2023.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Tsunematsu Yuta

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    Authorship:Principal investigator 

    Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

    CPF was discovered from a genetically engineered strain of a mushroom, Coprinopsis cinerea. CPF can enhance the growth of mycelium and facilitate fruiting body formation in the life cycle of this fungus through its affinity to bind environmental iron. This study explored the ubiquity of CPF's presence and its bioactive functions in basidiomycetes. Initially, we identified various Coprinopsis species harboring genes responsible for CPF biosynthesis, and Coprinus phlyctidosporus was determined to produce CPF. We achieved total synthesis of CPF, which enabled us to confirm its molecular structure and biological activities unambiguously. Furthermore, functional assessment of the CPF biosynthetic enzyme Cpf1 demonstrated its valuable attributes, including substantial substrate adaptability and the ability to generate diverse molecules.

  4. Integrating Computational and Experimental Approaches for New Paradigms in Molecule Synthesis

    Grant number:19KK0150  2019.10 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))  Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

    Watanabe Kenji

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    Authorship:Coinvestigator(s) 

    Here we show the crystal structure of CghA&#8211;product complex at 2.0 resolution. Our result provides the second structural information of eukaryotic Diels&#8211;Alderases and adds yet another fold to the family of proteins reported to catalyse [4+2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exon adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions, and paves the way toward developing enzymes potentially useful in various bio/synthetic applications.

  5. Engineered production and biosynthesis of mushroom-derived natural products from the encrypted biosynthetic gene

    Grant number:17K15265  2017.4 - 2019.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)  Grant-in-Aid for Young Scientists (B)

    Tsunematsu Yuta

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    Mushrooms are used not only as food but also as traditional natural medicines (Kampo) and health supplements, and they have been shown to have various effects on the human body. In this study, we investigated the biosynthesis of mushroom-derived bioactive substances at the gene, enzyme, and compound levels, respectively. Increased production of bioactive substances could be accomplished by overexpression of mushroom genes. On the other hand, it was confirmed that the novel functional molecule obtained by the gene disruption method is a compound involved in the growth and differentiation of the mushroom. It may be a new tool that can be used for mushroom cultivation and breeding.

  6. TGF-βシグナル伝達を阻害する新規異常トリプトファン含有天然物の作用機序解明

    Grant number:10J02153  2010 - 2011

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    恒松 雄太

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    Authorship:Principal investigator 

    Grant amount:\1400000 ( Direct Cost: \1400000 )

    我々が見出したStreptomyces属放線菌が生産する新規化合物tryptopeptin類は、既存の報告にはない新たな作用機序をもつTGF-βシグナル伝達経路阻害剤であると考えられ、新しい抗がん剤のリード化合物として期待される。本化合物は異常アミノ酸からなるペプチド骨格を有し、そのC末端側にはトリプトファン残基が2炭素増炭し、さらにエポキシケトンが形成されたユニークな化学構造を有する。本研究では、未決定であったtryptopeptin Aおよびtryptopeptin Bを全合成することにより絶対立体構造を解明した。続いて、得られた天然物および、合成誘導体をもとに構造活性相関を精査したところ、本化合物のTGF-βシグナル伝達経路阻害活性にはC末端のエポキシケトンが重要な役割を担うことが示唆された。続いて、本化合物の細胞内標的分子を突き止めるためにケミカルプローブを合成した。作成した蛍光化tryptopeptin Aを哺乳動物細胞へ処理したところ、特定のオルガネラへの蛍光の集積が認められたことから特異な標的分子が存在することが示唆された。一方、本研究では天然からは微量成分としてしか得られなかったtryptopeptin Aの全合成を達成し、効率的に本化合物を供給する方法を見出した。今後、本化合物を用いて更なる構造活性相関と詳細な作用機序解明、および動物実験などによる薬剤としての開発が期待される。

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